Cryoprotecting agent, cryoprotecting and cryopreserved compositions, uses thereof, and methods of cryopreservation

ABSTRACT

The present invention relates to a cryoprotecting agent comprising a cryprotectant being one or more of: dextrin, dextran, isomaltooligosaccharide and derivatives thereof, cryoprotecting and cryopreserved compositions, uses thereof, and methods of cryopreservation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/EP2013/075128, filed Nov. 29, 2013, which International Applicationwas published by the International Bureau in English on and which claimsthe benefit of U.S. Provisional Patent Application No. 61/731,849, filedNov. 30, 2012 and U.S. Provisional Patent Application No. 61/740,211,filed Dec. 20, 2012, which applications claim priority to EP ApplicationNo. 12195033.1, filed Nov. 30, 2012; EP Application No. 12198598.0,filed Dec. 20, 2012; and EP Application No. 13180869.3, filed Aug. 19,2013, all of which are hereby incorporated herein in their entirety byreference.

FIELD OF THE INVENTION

The present invention relates to a cryoprotectant, a cryoprotectingagent, cryoprotecting compositions, cryopreserved compositions, usesthereof, and methods of cryopreservation.

BACKGROUND OF THE INVENTION

Cryopreservation of viable biological samples such as cells, tissues ororgans, which have been harvested from a donor source, is of greatimportance and utility in the scientific and medical communities.Cryopreservation is generally a process where a sample, for examplecells or tissue, is preserved by cooling to sub-zero temperatures,typically 77 K (=196° C., the boiling point of liquid nitrogen). Atthese low temperatures, any biological activity, including thebiochemical reactions that would cause cell death, is effectivelystopped. Cryopreservation techniques are routinely used for long-timepreservation of water-bearing or aqueous materials such as cells andtissues of plants and animals. It is known that upon freezing thesematerials, ice crystals form, resulting in uneven concentrations ofsolutes and contaminants excluded by water molecules, called ‘freezeconcentration’. In order for the cells or tissues to be preserved,cryoprotectant solutions are typically used to prevent damage due tofreezing during the cooling or thawing process. For cryopreservation tobe useful, the preserved sample should retain it's integrity andviability to a reasonable level at the time of harvest. Thus, theprocess of preserving the sample should preferably not, in itself,severely damage or destroy for example the cells or tissue architecture.

In conventional cryopreservation techniques, the sample is harvested,placed in a storage solution, and then preserved by freezing. When thesample is to be used, it is thawed, and for example cells taken fromhuman donor sources are brought back to the normal human bodytemperature (i.e., approximately 37° C.) and then placed in a cellculture medium. Cryopreservation protocols subject the cells to amultitude of stresses and insults throughout the process of cellharvesting, freezing, and thawing. These stresses and insults can causeirreversible damage to the cell.

Dextran has been used as a cryoprotective agent for human, animal andplant cells (Odavic, R. et al. Experientia 36, 1122 (1980),Ashwood-Smith, M. J. et al. Cryobiology 9, 441 (1972) and Echlin, P. etal. J. Microsc. (Oxford) 110, 239 (1977)). A mixture of 5% methylsulphoxide and 9% Dextran 70 was found to afford optimal cryoprotectionof human bone marrow committed stem cells (Dextran, Handbook fromAmersham BioSciences, 18-1166-12, Edition AA, page 35). Dextran,glycerol and dimethyl sulfoxide (DMSO), alone or in combination, havebeen investigated for cryoprotection of human bone marrow cells (Odavic,R. et al. Experientia 36, 1122 (1980)). A significantly betterprotection against freezing injury was obtained by 9% Dextran 70 incombination with 3 or 5% DMSO, and also with 5 or 10% DMSO alone, thanwith either 15% glycerol or 9% dextran with 1% DMSO. Dextran 40 is known(Proc. Nati. Acad. Sci. USA, Vol. 92, pp. 10119-10122, October 1995,Medical Sciences) for cryopreservation of placental/umbilical cord bloodin a combination of 50% DMSO in 5% (w/v) Dextran 40.

Shu Guowei et al describes in Advanced Materials Research, Trans TechPublications Ltd., Vol. 328 (2012), pp 454-457 the effect offructo-oligosaccharide, isomalto-oligosaccharide, inulin andxylo-oligosaccharide on survival of B. bifidium during freeze-drying.Kwan Hwa Park et al. describes in Database CA, XP 002698458 acryoprotectant containing fructooligosaccharide, isomaltooligosaccharideor galactooligosaccharide for surimi. In J. Korean Soc. Food Sci. Nutr.,Vol. 30(3) (2001), pp 565-568, the effects of a cryoprotectant offructooligosaccharide, isomaltooligosaccharide andgalactooligosaccharide on beef protein is described.

Conventional cryoprotectants are glycols (alcohols containing at leasttwo hydroxyl groups), such as ethylene glycol, propylene glycol, andglycerol. Ethylene glycol is commonly used as automobile antifreeze, andpropylene glycol has been used to reduce ice formation in ice cream.Dimethyl sulfoxide (DMSO) is also regarded as a conventionalcryoprotectant. Glycerol and DMSO have been used for decades bycryobiologists to reduce ice formation in sperm and embryos that arepreserved in liquid nitrogen (−196° C.). Among these knowncryoprotecting agents, DMSO is considered the most effective andfrequently adopted, but it is physiologically toxic and known to causehigh blood pressure, nausea and vomiting if transfused to a recipientwith the cells or for personnel handling it unless precautions aretaken. Cox et al (Cell Tissue Bank (2012) 13:203-215) identified by aretrospective review of published literature several hundred adversereactions (e.g. nausea, chills, cardiac arrhythmias, neurologicalsymptoms and respiratory arrest) associated with the transplantation ofstem cells cryopreserved with dimethyl sulfoxide. Further, the toxicityof DMSO tends to debilitate the cells' survival rates and/or functions,including genomic alterations after the thawed cells are cultured ortransfused into a recipient's body. The toxicity of DMSO thus alsoaffects how long the cells may be exposed to DMSO during handling.

Thus, there is still a need for a cryoprotecting agent for protecting asample such as a biological sample during freezing as a replacement forother cryoprotectants such as DMSO or as a supplement to such othercryoprotectants to reduce the concentration needed thereof, preferablyto non-toxic concentrations, which at the same time have the necessaryprotective effects and low toxicity.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide acryoprotectant as a replacement for other cryoprotectants such as DMSOor as a supplement to such other cryoprotectants to reduce theconcentration thereof, preferably to non-toxic concentrations, whichcryoprotectant have the necessary protective effects with regard topreserving as much functionality of the cryopreserved sample duringcryopreserving. It is a further object of embodiments of the inventionto provide a cryoprotectant having a low toxicity for the personnelhandling the cryoprotectant and for the biological samples, whereby thetime that the sample can be in contact with the cryoprotectant withoutbeing damaged is prolonged, the necessity for washing of samples isreduced, and preferably, if desired, making it possible to return thesample to where it was taken from or to a recipient without having toseparate the sample from the cryoprotectant. It is a further object ofembodiments of the invention to provide a cryoprotectant which iseffective as a cryoprotectant for a sample selected from the groupconsisting of organs, cells and tissues such as selected from the groupconsisting of mammalian organs, mammalian cells, and mammalian tissues.It is a further object of embodiments of the invention to provide acryoprotectant which is effective as a cryoprotectant for a sample to betransplanted such as an organ, cells or tissues. It is a further objectof embodiments of the invention to provide a cryoprotectant which iseffective as a cryoprotectant of for example cells and result in anacceptable viability of said cells. It is a further object ofembodiments of the invention to provide a cryoprotectant which iseffective as a cryoprotectant of for example organs and result in anacceptable physical functionality of said organs. It is a further objectof embodiments of the invention to provide a cryoprotectant which iseffective as a cryoprotectant of for example tissues and result in anacceptable physical functionality of said tissues.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that a cryoprotecting agentcomprising one or more cryoprotectants selected from the groupconsisting of dextrin, dextran, isomaltooligosaccharide, and derivativesthereof, and a) wherein said cryoprotecting agent comprises at least 1%w/w of one or more of isomaltooligosaccharide and derivatives thereofhaving a weight average molecular weight (M_(w)) of between 300 and1,650 Da based on the total weight of dextrin, dextran,isomaltooligosaccharide, and derivatives thereof in said agent, or b)wherein said cryoprotectant has a weight average molecular weight(M_(w)) of between 300 and 9,500 Da such as between 300 and 7,500 Da, orc) wherein said cryoprotecting agent comprises at least 1% w/w of one ormore of isomaltooligosaccharide and derivatives thereof having a weightaverage molecular weight (M_(w)) of between 300 and 1,650 Da based onthe total weight of dextrin, dextran, isomaltooligosaccharide andderivatives thereof in said agent, and said cryoprotectant has a weightaverage molecular weight (M_(w)) of between 300 and 9,500 Da, is veryuseful as a cryoprotectant. Compared to the previously used dextranmaterials of higher molecular weight such as Dextran 40 (40,000 Da) andDextran 70 (70,000 Da), the cryoprotecting agent comprising abovedescribed cryoprotectant has a lower viscosity due to the lowermolecular weight and may thus be pre-prepared in a high concentrationmaking it possible to add a sample already in a solution and stillobtain a composition comprising both cryoprotectant and sample in aconcentration suitable for cryopreservation. Furthermore, molecules withlower molecular weight are generally less immunogenic than moleculeswith high molecular weight. Dextran 1 is known as a hapten inhibitorthat reduces the risk for anaphylactic reactions when administeringdextran and is thus used as pre-injection before injection of dextranswith higher molecular weight such as Dextran 40 (40,000 Da) and Dextran70 (70,000 Da). Dextran 1 has also been documented in studies by Richteret al (Int. Arch. Allergy 43:252-268 (1972) and Int. Arch. Allergy41:826-844 (1971)) to have a very low immunological potential in humans.Furthermore, it has been shown in the examples that cryopreservationwith the cryoprotectant as described herein provides a better protectionof functionality after cryopreservation measured as viability of thetested cells than with dextrans having a higher molecular weight.

So, in a first aspect the present invention relates to a cryoprotectingagent comprising one or more cryoprotectants selected from the groupconsisting of dextrin, dextran, isomaltooligosaccharide, and derivativesthereof, and

-   -   a) wherein said cryoprotecting agent comprises at least 1% w/w        of one or more of isomaltooligosaccharide and derivatives        thereof having a weight average molecular weight (M_(w)) of        between 300 and 1,650 Da based on the total weight of dextrin,        dextran, isomaltooligosaccharide, and derivatives thereof, or    -   b) wherein said cryoprotectant has a weight average molecular        weight (M_(w)) of between 300 and 9,500 Da such as between 300        and 7,500 Da or,    -   c) wherein said cryoprotecting agent comprises at least 1% w/w        of one or more of isomaltooligosaccharide and derivatives        thereof having a weight average molecular weight (M_(w)) of        between 300 and 1,650 Da based on the total weight of dextrin,        dextran, isomaltooligosaccharide and derivatives thereof in said        cryoprotecting agent, and said cryoprotectant has a weight        average molecular weight (M_(w)) of between 300 and 9,500 Da.

In a further aspect, the present invention relates to a cryoprotectingagent comprising a cryoprotectant selected from the group consisting ofisomaltooligosaccharide and derivatives thereof having a weight averagemolecular weight (M_(w)) of between 300 and 1,650 Da, such as between850 and 1,650 Da.

In a further aspect, the present invention relates to the use of acryoprotectant as described herein for cryopreserving a sample, whereinsaid sample is selected from the group consisting of organs, cells andtissues.

In a further aspect, the present invention relates to a cryopreservationcomposition comprising a cryoprotecting agent as described herein, whichcryopreservation composition further comprises a sample to becryopreserved, wherein said sample is selected from the group consistingof organs, cells and tissues.

In a further aspect, the present invention relates to a cryopreservedcomposition comprising a cryoprotecting agent as described herein, whichcryopreserved composition further comprises a sample which has beencryopreserved, wherein said sample is selected from the group consistingof organs, cells and tissues.

In a further aspect, the present invention relates to a method ofcryopreserving a sample, comprising the steps of bringing a sample to becryopreserved into contact with a cryoprotecting agent as describedherein to obtain a cryopreservation composition and subsequentlyreducing the temperature of the cryopreservation composition to acryopreservation temperature, wherein said sample is selected from thegroup consisting of organs, cells and tissues.

In a further aspect, the present invention relates to a method ofcryopreserving a cryopreservation composition as described herein bybringing the composition to a cryopreservation temperature.

In a further aspect, the present invention relates to the use of acryoprotecting agent as described herein for cryopreserving a sample,wherein said sample is selected from the group consisting of organs,cells and tissues.

In a further aspect, the present invention relates to the use of acryoprotecting agent as described herein for cryopreserving a sample fortransplantation.

In a further aspect, the present invention relates to the use of acryopreservation composition as described herein for cryopreserving asample by reducing the temperature of said composition to acryopreservation temperature, wherein said sample is selected from thegroup consisting of organs, cells and tissue.

LEGENDS TO THE FIGURE

FIG. 1 shows survival of NHDFs after thawing and viability after 1.passage in respectively 1) 10% DMSO, 2) 8% isomaltooligosaccharide 1(ISOM) and 2% DMSO, 3) 8% isomaltooligosaccharide 1 (ISOM) and 4) DMEMas described in Example 2.

FIG. 2 shows survival of NHDFs after thawing and viability after 1.passage in respectively 1) 10% DMSO, 2) 8% hydrogenatedisomaltooligosaccharide 1 (H-ISOM) and 2% DMSO, 3) 8% hydrogenatedisomaltooligosaccharide 1 (H-ISOM) and 4) DMEM as described in Example2.

FIG. 3 shows survival of NHEKs after thawing and viability after 1.passage in respectively 1) 10% DMSO, 2) 8% isomaltooligosaccharide 1(ISOM) and 2% DMSO, 3) 8% isomaltooligosaccharide 1 (ISOM) and 4) DMEMas described in Example 3.

FIG. 4 shows survival of NHEKs after thawing in respectively 1) 10%DMSO, 2) 8% hydrogenated isomaltooligosaccharide 1 (H-ISOM) and 2% DMSO,3) 8% hydrogenated isomaltooligosaccharide 1 (H-ISOM) and 4) DMEM asdescribed in Example 3.

FIG. 5 shows survival of MSCs after thawing and viability after 1.passage in respectively 1) 10% DMSO, 2) 8% isomaltooligosaccharide 1(ISOM) and 2% DMSO, 3) 8% hydrogenated isomaltooligosaccharide 1(H-ISOM) and 2% DMSO, 4) 8% isomaltooligosaccharide 1 (ISOM), 5) 8%hydrogenated isomaltooligosaccharide 1 (H-ISOM) and 6) DMEM as describedin Example 4.

FIG. 6 shows survival of Human iPS cells in PluriPro growth medium afterthawing and cryopreservation in respectively Growth medium+10% DMSO,Growth medium+5% DMSO, Growth medium+10% DMSO+2% isomaltooligosaccharide1 (ISOM), Growth medium+10% DMSO+4% isomaltooligosaccharide 1 (ISOM),Growth medium+10% DMSO+8% isomaltooligosaccharide 1 (ISOM), Growthmedium+5% DMSO+2% isomaltooligosaccharide 1 (ISOM), Growth medium+5%DMSO+4% isomaltooligosaccharide 1 (ISOM), Growth medium+5% DMSO+8%isomaltooligosaccharide 1 (ISOM), Growth medium+2%isomaltooligosaccharide 1 (ISOM), Growth medium+4%isomaltooligosaccharide 1 (ISOM), Growth medium+8%isomaltooligosaccharide 1 (ISOM), and Growth medium without anycryoprotectant as described in Example 7.

FIG. 7 shows survival of Human iPS cells in PluriPro growth medium afterthawing and cryopreservation in respectively Growth medium+10% DMSO,Growth medium+5% DMSO, Growth medium+10% DMSO+2% hydrogenatedisomaltooligosaccharide 1 (H-ISOM), Growth medium+10% DMSO+4%hydrogenated isomaltooligosaccharide 1 (H-ISOM), Growth medium+10%DMSO+8% hydrogenated isomaltooligosaccharide 1 (H-ISOM), Growthmedium+5% DMSO+2% hydrogenated isomaltooligosaccharide 1 (H-ISOM),Growth medium+5% DMSO+4% hydrogenated isomaltooligosaccharide 1(H-ISOM), Growth medium+5% DMSO+8% hydrogenated isomaltooligosaccharide1 (H-ISOM), Growth medium+2% hydrogenated isomaltooligosaccharide 1(H-ISOM), Growth medium+4% hydrogenated isomaltooligosaccharide 1(H-ISOM), Growth medium+8% hydrogenated isomaltooligosaccharide 1(H-ISOM) and Growth medium without any cryoprotectant as described inExample 8.

FIG. 8 shows survival of MSCs after thawing and proliferation after 3days in respectively 1) 10% DMSO, 2) “CRYO” which designates 8%isomaltooligosaccharide 1, Dextran Mw10.000 or Dextran Mw 40.000respectively, each combined with 5% DMSO, 3) “CRYO” which designates 8%isomaltooligosaccharide 1, Dextran Mw 10.000 or Dextran Mw 40.000respectively, each combined with 1% DMSO, 4) “CRYO” which designates 8%isomaltooligosaccharide 1, Dextran Mw 10.000 or Dextran Mw 40.000respectively and 5) DMEM as further described in Example 9.

FIG. 9 shows survival of MSCs after thawing in respectively 1) 10% DMSO,2) 2% DMSO, 3) 8% isomaltooligosaccharide Mw 1500 (ISOM) and 2% DMSO, 4)8% isomaltooligosaccharide Mw 1500 (ISOM), and 5) DMEM as furtherdescribed in Example 10.

FIG. 10 shows viability of CD34+ hematopoietic stem cell followingcryopreservation with DMSO, isomaltooligosaccharide 1 or hydrogenatedisomaltooligosaccharide 1 as further described in example 11.

FIG. 11 shows viability of adipose-derived stromal/stem cells (ASC's)following cryopreservation with DMSO, isomaltooligosaccharide 1 orhydrogenated isomaltooligosaccharide 1 as further described in example12.

DETAILED DISCLOSURE OF THE INVENTION Definitions

As used herein, cryopreservation means a process where a sample ispreserved by cooling to sub-zero temperatures, including vitrificationtechnology in which the cooling rate is faster than a conventionalcryopreservation procedure. At such low temperatures activity such asbiological activity, including the biochemical reactions that wouldcause for example cell death, are reduced, and the chemicalstructure/function of for example proteins/glyco-proteins orlipoproteins are preserved. If cryoprotectant solutions are not used,the samples being preserved are likely to be damaged due to freezingduring the cooling or thawing process.

In one preferred aspect, cryopreservation means a process where samplesare preserved by cooling to sub-zero temperatures, typically 77 K (=196°C., the boiling point of liquid nitrogen). At these low temperatures,any biological activity, including the biochemical reactions that wouldcause cell death, is effectively stopped.

As used herein, the term “cryopreservation temperature” designates atemperature of from sub-zero to −196° C., such as from −50° C. to −196°C., such as from −80° C. to −196° C., such as a temperature below −55°C., such as below −60° C., such as below −65° C., such as below −70° C.,such as below −75° C., such as below −80° C., such as below −85° C.,such as below −90° C., such as below −95° C., such as below −100° C.,such as below −105° C., such as below −110° C., such as below −115° C.,such as below −120° C., such as below −125° C., such as below −130° C.,such as below −135° C., such as below −140° C., such as below −145° C.,such as below −150° C., such as below −155° C., such as below −160° C.,such as below −165° C., such as below −170° C., such as below −175° C.,such as below −180° C., such as below −185° C., such as below −190° C.

As used herein, the term “sample” means any kind of material to becryopreserved such as organs, cells, or tissue. In one aspect, a sampleis selected from the group consisting of organs, cells, tissue andblood. In one aspect, a sample is selected from the group consisting oforgans, cells, and tissue, such as selected from the group consisting ofmammalian organs, mammalian cells and mammalian tissues. In one aspect,the term “sample” does not comprise the human body at the various stagesof its formation and development. In a further aspect, the presentinvention relates to the use of a cryoprotecting agent as describedherein for cryopreserving a sample for transplantation. In one aspect,the sample is selected from the group consisting of mammalian organs,mammalian cells and mammalian tissues for transplantation.

As used herein, the term “cells” comprises any type of cells such assomatic cells including all kind of cells in tissue or organs, stemcells including all types of totipotent stem cells, pluripotent stemcells, multipotent stem cells and progenitor cells; oocytes;spermatozoa; and germ cells. The cells may be in isolated form or in anot isolated form such as in the form of a cell-containing bodily fluid,a tissue or organ.

As used herein, the term “cell-containing bodily fluids” comprises anycell-containing bodily fluid such as for example below defined blood,amniotic fluid, semen, cerebrospinal fluid, bone marrow aspirates andmenstrual fluid.

As used herein, the term “blood” comprises any blood containing fluidsuch as umbilical cord blood, peripheral blood, and mobilized blood.

As used herein, the term “tissue” or “tissues” comprises any tissue typecomprising any kind of cell type and combinations thereof, includingovarian tissue, testicular tissue, umbilical cord tissue, placentaltissue, connective tissue, cardiac tissue, tissues from muscle,cartilage and bone, endocrine tissue, and neural tissue. The term“tissue” or “tissues” also comprise adipose tissue or dental pulptissue.

As used herein, the term “organ” comprises for example lung, liver,kidney, heart, ovaries and pancreas. The term “organ” also compriseumbilical cord.

As used herein, the term “functional after cryopreservation” in relationto a sample means that the sample such as organs, tissue or cells aftercryopreservation retains an acceptable and/or desired function aftercryopreservation. In one aspect, the sample after cryopreservationretains all its function. In another aspect, the sample, such as cells,at least retains 50% of the desired function, such as at least 60% ofthe desired function, such as at least 70% of the desired function, suchas at least 80% of the desired function, such as at least 90% of thedesired function, such as at least 95% of the desired function, such as100% of the desired function, As an example with regards to cells animportant function to be preserved is the viability of the cells. Asanother example with regards to organs an important function to bepreserved is the physiological function of such organ, e.g. for a heartthe pumping function. As another example with regards to tissue animportant function to be preserved is the ability of such tissue tointegrate with surrounding tissue (e.g. skin) in the case oftransplantation.

The viability of cells after cryopreservation may be measured by usingthe Nucleocounter system in which dead cells are measured by incubatingthe cell sample with the DNA binding dye, propidium iodide, which onlyresults in detectable measurement from dead cells as shown in theexamples. The viability is given as a percentage of living cells in thepopulation that are being analyzed. The proliferative rate of a cellsample after cryopreservation can be analysed using the colorimetricassay, MTT.

As used herein, the term “banking” means any storage of a sample forfuture use.

As used herein, the term “clinical banking method” means any storage ofa sample relating to clinical treatment of a mammal such as a humanbeing.

As used herein, the term “marrow banking method” means storage of amarrow sample such as bone marrow aspirates and related bodily fluid, orcells isolated from marrow.

As used herein, the term “dental pulp tissue banking method” meansstorage of a dental pulp tissue sample such as cells isolated fromdental pulp tissue.

As used herein, the term “fat tissue banking method” means storage of afat tissue sample such as cells isolated from fat tissue. In the presentcontext the term and in the following, “umbilical cord banking method”means storage of umbilical cord blood, tissues related to umbilical cordor cells isolated from umbilical cord blood or tissue.

In the present context the term and in the following, “mobilizedperipheral blood banking method” means storage of peripheral blood aftermobilizing with agents that for example releases blood stem cells intothe circulation.

As used herein the term, “reproduction banking method” means storage ofany sample relating to reproduction such as semen, oocytes, spermatozoa,fertilized eggs etc.

The atomic mass unit Dalton (symbol: Da) is the standard unit used forindicating mass on an atomic or molecular scale (atomic mass). It isdefined as one twelfth of the remaining mass of an unbound neutral atomof carbon-12 in its nuclear and electronic ground state.

As used herein, the term “weight average molecular weight” (M_(w)) isdefined as:

$M_{w} = \frac{\sum\limits_{i = 1}^{i = p}\;{g_{i}M_{i}}}{\sum\limits_{i = 1}^{i = p}\; g_{i}}$wherein g_(i) is the fraction of molecules having the molecular weightM_(i). The possible values of M make up a set of numbers with discretevalues labelled M_(i), defining p.

As used herein, the term “number average molecular weight” is definedas:

$M_{n} = {\frac{\sum\limits_{i = 1}^{i = p}\;{\left( {g_{i}\text{/}M_{i}} \right)M_{i}}}{\sum\limits_{i = 1}^{i = p}\;\left( {g_{i}\text{/}M_{i}} \right)} = \frac{\sum\limits_{i = 1}^{i = p}\;{N_{i}M_{i}}}{\sum\limits_{i = 1}^{i = p}\; N_{i}}}$

wherein N_(i) is the fraction of molecules having the molecular weightM_(i), g_(i) is the fraction of molecules having the molecular weightM_(i). The possible values of M make up a set of numbers with discretevalues labelled M_(i), defining p.

As used herein, the term polydispersity (Pd) is calculated byM_(w)/M_(n)=Pd.

As used herein, the term “Dextran” followed by a number such as “Dextran1”, “Dextran 40” and “Dextran 70” follows the Pharmacopoeialabbreviation for Dextran X meaning that the weight average molecularweight of the dextran is approximately X kDA. Thus Dextran 1 means adextran having a weight average molecular weight of 850-1,150 Da.Isomaltooligosaccharide 1, and hydrogenated isomaltooligosaccharide 1are named similarly. Isomaltooligosaccharide 1 means thus a mixture ofisomaltooligosaccharides having a weight average molecular weight of850-1,150 Da conforming to the EP and USP Monographs for Dextran 1.Isomaltooligosaccharide 1 is also named pentaisomaltose in thisapplication. Hydrogenated isomaltooligosaccharide 1 means a mixture ofhydrogenated isomaltooligosaccharides, where theisomaltooligosaccharides conforms to the EP and USP Monographs forDextran 1. Hydrogenated isomaltooligosaccharide 1 is also namedpentaisomaltoside in this application.

As used herein, the term “dextran-based isomaltooligosaccharide” meansan isomaltooligosaccharide having a weight average molecular weight(M_(w)) of between 300 and 1,650 Da, such as between 850 and 1,650 Daand obtained from hydrolysed dextran such as by hydrolysis of lowmolecular weight dextran.

As used herein, the term “cryoprotectant” means a substance which forexample in an appropriate solution is used to protect a sample fromfreezing damage. Examples of known cryoprotectants are for example DMSO,polyols etc.

As used herein, the term “sterile” means free from living germs,microorganisms and other organisms capable of proliferation.

As used herein, the term “substantially free of DMSO” means DMSO in anamount less than 0.01 w/w %.

As used herein, “C₁₋₁₀alkyl” is a hydrocarbon being a straight-chain orbranched C₁₋₁₀alkyl such as a straight-chain or branched C₁₋₆alkyl.Examples are methyl, ethyl, 1-propyl, 2-propyl, isopropyl, 1-butyl,2-methyl-1-propyl, 2-butyl, 1-pentyl, 3-pentyl, 2-methyl-2-butyl, and3-methyl-2-butyl.

As used herein, “carboxyC₁₋₁₀alkyl” means —C₁₋₁₀alkylCOOH. An example iscarboxymethyl (CM) (—CH₂COOH).

As used herein, the term “DEAE” means diethylaminoethyl.

Cryoprotecting Agent

Described herein is a cryoprotecting agent comprising a cryoprotectantbeing one or more selected from the group consisting of dextrin,dextran, isomaltooligosaccharide, and derivatives thereof, and

-   -   a) wherein said cryoprotecting agent comprises at least 1% w/w        of one or more of isomaltooligosaccharide and derivatives        thereof having a weight average molecular weight (M_(w)) of        between 300 and 1,650 Da such as a weight average molecular        weight (M_(w)) of between 850 and 1,650 Da based on the total        weight of dextrin, dextran, isomaltooligosaccharide, and        derivatives thereof in said cryoprotecting agent, or    -   b) wherein said cryoprotectant has a weight average molecular        weight (M_(w)) of between 300 and 9,500 Da such as of between        300 and 7,500 Da, or    -   c) wherein said cryoprotecting agent comprises at least 1% w/w        of one or more of isomaltooligosaccharide and derivatives        thereof having a weight average molecular weight (M_(w)) of        between 300 and 1,650 Da such as between 850 and 1,650 Da based        on the total weight of dextrin, dextran, isomaltooligosaccharide        and derivatives thereof in said cryoprotecting agent, and said        cryoprotectant has a weight average molecular weight (M_(w)) of        between 300 and 9,500 Da.

In one aspect, said cryoprotectant is selected from the group consistingof dextran, isomaltooligosaccharide, and derivatives thereof,

The molecular weight of dextran and dextrin, and/or a derivative thereofis typically determined by means of gel permeation chromatography (GPC)using for example GPC columns of the type polyether hydroxylated gels.The calibration may be performed as described in the EuropeanPharmacopoeia 7^(th) Edition for dextran and using the iterativemathematical method as described in the European Pharmacopoeia 7^(th)Edition, volume 2, pages 1816-1817, for dextran.

The molecular weight of isomaltooligosaccharide and/or a derivativethereof such as hydrogenated isomaltooligosaccharide, is typicallydetermined by means of gel permeation chromatography (GPC). Thestationary phase in the column system may be dextran covalently bound tohighly cross-linked porous agarose beads, allowing resolution ofoligosaccharides in the molecular mass range 180-3000 Da. Themeasurement is made in accordance with the European Pharmacopoeia 7^(th)Edition, volume 1, page 60-61.

When said cryoprotectant is electrical neutral the weight averagemolecular weight (M_(w)) of said cryoprotectant is preferably measuredby GPC. When measuring weight average molecular weight (M_(w)) of acryoprotectant carrying an electrical charge, the weight averagemolecular weight (M_(w)) is calculated based on the molecular weight ofthe electrical neutral starting material, and the substitution degree ofthe charged cryoprotectant. Each glucose unit in the uncharged startingmaterial can be substituted with between 1 and 3 substituents. UsingDEAE as an example of a substituent the skilled person may for examplemeasure the nitrogen content (e.g. using Kjeldahl analysis) to calculatethe degree of substitution an thereafter calculate the molecular weightof the final product. If the substituent contains an acid group thedegree of substitution may for example be determined by the skilledperson by titration, and thereafter the final molecular weight may becalculated.

Dextrin, dextran, and isomaltooligosaccharide all comprise repeatingD-glucose units. Dextrans are a family of neutral branchedpolysaccharides consisting predominantly of an α-(1→6) linked D-glucoseas further described in the following. Dextrins are mixtures of polymersof D-glucose units linked by α-(1→4) or α-(1→6) glycosidic bonds asfurther described in the following. Isomaltooligosaccharide is a mixtureof glucose oligomers with α-D-(1,6)-linkages (typically fewer than 10D-glucose units, suitably between 3-6 glucose units) and typically hasan average weight molecular weight between 300 and 1,650 Da, such asbetween 500 and 1,650 Da, such as between 850 and 1,650 Da, or such asbetween 850 Da and 1150 Da. In one aspect, the weight fraction ofisomaltooligosaccharides having less than 3 glucose units is less than15% w/w. In one aspect, the weight fraction of isomaltooligosaccharideshaving more than 9 glucose units is less than 20% w/w such as less than15% w/w, such as less than 10% w/w. In a further aspect, the weightfraction of isomaltooligosaccharides having less than 3 glucose units isless than 15% w/w and the weight fraction of isomaltooligosaccharideshaving more than 9 glucose units is less than 20% w/w such as less than15% w/w, such as less than 10% w/w. The weight fraction may for examplebe determined as described in Preparation example 1 and 2 herein.

In one aspect, a derivative of dextrin, dextran, andisomaltooligosaccharide are selected from the group consisting ofhydrogenated isomaltooligosaccharide, hydrogenated dextran, hydrogenateddextrin, oxidised isomaltooligosaccharide, oxidised dextran, oxidiseddextrin, ester of dextrin, ester of dextran, ester ofisomaltooligosaccharide, ether of dextrin, ether of dextran, ether ofisomaltooligosaccharide, and partly hydrogenated/oxidised dextrin,partly hydrogenated/oxidised dextran and partly hydrogenated/oxidisedisomaltooligosaccharide, and derivatives thereof. In one aspect, aderivative of isomaltooligosaccharide is selected from the groupconsisting of hydrogenated isomaltooligosaccharide, oxidisedisomaltooligosaccharide, ester of isomaltooligosaccharide, ether ofisomaltooligosaccharide, and partly hydrogenated/oxidisedisomaltooligosaccharide, and derivatives thereof.

Below is a schematic overview (Table A) of examples of the differentsynthesises and starting materials for the above described derivativesof dextrin, dextran, and isomaltooligosaccharide:

TABLE A Starting material/ Hydro- Esteri- Etheri- Synthesis genationOxidation* fication fication Dextran x x x x Isomaltooligo- x x x xsaccharides Dextrin x x x x Hydrogenated dextran x x x Hydrogenated x xx isomaltooligo- saccharides Hydrogenated Dextrin x x x Oxidated dextranx x x Oxidated isomaltooligo- x x x saccharides Oxidated dextrin x x x*in one aspect, the synthesis may be a partly oxidation andhydrogenation method to obtain partly oxididised and hydrogenatedderivatives for example as described in U.S. Pat. No. 6,977,249

In one aspect, the type of said ether of dextrin, ether of dextran andether of isomaltooligosaccharide is selected from the group consistingof ethers having a functional group R. R is selected from the groupconsisting of C₁₋₁₀alkyl such as C₁₋₆alkyl such as methyl (—CH₃) andethyl (—C₂H₅), carboxyC₁₋₁₀alkyl such as carboxymethyl (—CH₂COOH),hydroxy C₁₋₁₀alkyl such as 2-hydroxyethyl (—C₂H₄OH), 2-hydroxypropyl(—CH₂CHOHCH₃), 2-hydroxyalkyl (—CH₂CHOH(CH2)_(n)CH₃ wherein n is 1-10),3-chloro-2-hydroxypropyl (—CH₂CHOHCH₂Cl), 2-diethylaminoethyl(—C₂H₄N(C₂H₅)₂), 3-amino-2-hydroxypropyl (—CH₂CHOHCH₂NH₂),3-dimethylalkylammonium-2-hydroxypropyl (—CH₂CHOHCH₂N′(CH₃)₂R wherein Ris C₁₋₁₀alkyl), polyethyleneglycol cetyl (—CH₂CH₂O)₁₀C₁₆H₃₃), andpolyethyleneglycol stearyl (—CH₂CH₂O)₁₀C₁₈H₃₇).

In one aspect, said ether of dextrin, ether of dextran and ether ofisomaltooligosaccharide is respectively DEAE-dextrin, DEAE-dextran,DEAE-isomaltooligosaccharides. In one aspect, said ether ofisomaltooligosaccharide is DEAE-isomaltooligosaccharide. In one aspect,said ether of dextrin, ether of dextran and ether ofisomaltooligosaccharide is respectively carboxyC₁₋₁₀alkyl-dextrin,carboxyC₁₋₁₀alkyl-dextran, carboxyC₁₋₁₀alkyl-isomaltooligosaccharide. Inone aspect, said ether of isomaltooligosaccharide iscarboxyC₁₋₁₀alkyl-isomaltooligosaccharide,

In one aspect, a derivative of dextrin, dextran, andisomaltooligosaccharide are selected from the group consisting ofhydrogenated isomaltooligosaccharide, hydrogenated dextran andhydrogenated dextrin. In one aspect, a derivative ofisomaltooligosaccharide is hydrogenated isomaltooligosaccharide.

In another aspect, a derivative of dextrin, dextran, andisomaltooligosaccharide are selected from the group consisting ofoxidised isomaltooligosaccharide, oxidised dextran and oxidised dextrin.In one aspect, a derivative of isomaltooligosaccharide is oxidisedisomaltooligosaccharide. In another aspect, a derivative of dextrin,dextran, and isomaltooligosaccharide are selected from the groupconsisting of oxidised/hydrogenated isomaltooligosaccharide,oxidised/hydrogenated dextran and oxidised/hydrogenated dextrin. In oneaspect, a derivative of isomaltooligosaccharide is oxidised/hydrogenatedisomaltooligosaccharide.

In another aspect, a derivative of dextrin, dextran andisomaltooligosaccharide are selected from the group consisting ofDEAE-dextrin, DEAE-dextran, DEAE-isomaltooligosaccharide,carboxyC1-10alkyl-dextrin, carboxyC1-10alkyl-dextran,carboxyC1-10alkyl-isomaltooligosaccharide, ester of dextrin, ester ofdextran and ester of isomaltooligosaccharide. In another aspect, aderivative of isomaltooligosaccharide is selected from the groupconsisting of DEAE-isomaltooligosaccharide,carboxyC1-10alkyl-isomaltooligosaccharide, and ester ofisomaltooligosaccharide.

In another aspect, a derivative of dextrin, dextran, andisomaltooligosaccharide are selected from the group consisting ofderivatives of hydrogenated dextrin, hydrogenated dextran, hydrogenatedisomaltooligosaccharides such as selected from the group consisting ofDEAE-hydrogenated dextrin, DEAE-hydrogenated dextran, DEAE-hydrogenatedisomaltooligosaccharides, carboxyC₁₋₁₀alkyl-hydrogenated dextrin,carboxyC₁₋₁₀alkyl-hydrogenated dextran, carboxyC₁₋₁₀alkyl-hydrogenatedisomaltooligosaccharide, ester of hydrogenated dextrin, ester ofhydrogenated dextran and ester of hydrogenated isomaltooligosaccharide.In another aspect, a derivative of isomaltooligosaccharide is selectedfrom the group consisting of derivatives of hydrogenatedisomaltooligosaccharides such as selected from the group consisting ofDEAE-hydrogenated isomaltooligosaccharides,carboxyC₁₋₁₀alkyl-hydrogenated isomaltooligosaccharide, and ester ofhydrogenated isomaltooligosaccharide. In another aspect, a derivative ofdextrin, dextran, and isomaltooligosaccharide are selected from thegroup consisting of derivatives of oxidised dextrin, oxidised dextranand oxidised isomaltooligosaccharides such as selected from the groupconsisting of DEAE-oxidised dextrin, DEAE-oxidised dextran,DEAE-oxidised isomaltooligosaccharides, carboxyC₁₋₁₀alkyl-oxidiseddextrin, carboxyC₁₋₁₀alkyl-oxidised dextran, carboxyC₁₋₁₀alkyl-oxidisedisomaltooligosaccharide, ester of oxidised dextrin, ester of oxidiseddextran and ester of oxidised isomaltooligosaccharide. In anotheraspect, a derivative of isomaltooligosaccharide is selected from thegroup consisting of derivatives of oxidised isomaltooligosaccharidessuch as selected from the group consisting of DEAE-oxidisedisomaltooligosaccharides, carboxyC₁₋₁₀alkyl-oxidisedisomaltooligosaccharide, and ester of oxidised isomaltooligosaccharide.

In yet a further aspect, the cryoprotectant is a hydrogenatedisomaltooligosaccharide, an oxidised isomaltooligosaccharide,DEAE-isomaltooligosaccharid, a carboxyC₁₋₁₀alkyl oxidisedisomaltooligosaccharide, an ester of isomaltooligosaccharide, an etherof isomaltooligosaccharide or a carboxyC₁₋₁₀alkylisomaltooligosaccharide.

In yet a further aspect, the cryoprotectant is carboxymethylisomaltooligosaccharide or carboxyethyl isomaltooligosaccharide.

In one aspect, the cryoprotectant is isomaltooligosaccharide such asisomaltooligosaccharide 1. In another aspect, the cryoprotectant ishydrogenated isomaltooligosaccharide such as hydrogenatedisomaltooligosaccharide 1.

By variation of the derivatisation, various degrees of substitution canbe achieved. Suitably, the degree of substitution lays in the range oneto three substituents to each glucose unit. In the case of for exampleDEAE-dextran, preferable approximately one charge group to three glucoseunits.

In one aspect, the cryoprotecting agent comprises a cryoprotectant beingone or more of: dextrin, dextran, isomaltooligosaccharide, andderivatives thereof, and wherein said cryoprotecting agent comprises atleast 1% w/w of one or more of isomaltooligosaccharide and derivativesthereof having a weight average molecular weight (M_(w)) of between 300and 1,650 Da such as having a weight average molecular weight (M_(w)) ofbetween 850 and 1,650 Da based on the total weight of dextrin, dextran,isomaltooligosaccharide, and derivatives thereof in said agent.

In a further aspect, the cryoprotecting agent comprises a cryoprotectantbeing one or more of: dextrin, dextran, isomaltooligosaccharide, andderivatives thereof, wherein said cryoprotectant has a weight averagemolecular weight (M_(w)) of between 300 and 9,500 Da, such as between300 and 7,500 Da, such as between 500 and 7,500 Da. In one aspect, saidcryoprotectant has a weight average molecular weight (M_(w)) of at most9500 Da, such as at most 9000 Da, such as at most 8000 Da, such as atmost 7000 Da, such as at most 6000 Da, such as at most 5000 Da, such asat most 4000 Da, such as at most 3000 Da, such as at most 2000 Da, suchas at most 1900 Da, such as at most 1800 Da, such as at most 1700 orsuch as at most 1,650 Da.

In yet a further aspect, the cryoprotecting agent comprises acryoprotectant being one or more of: dextrin, dextran,isomaltooligosaccharide, and derivatives thereof, and wherein saidcryoprotecting agent comprises at least 1% w/w of one or more ofisomaltooligosaccharide and derivatives thereof having a weight averagemolecular weight (M_(w)) of between 300 and 1,650 Da such as having aweight average molecular weight (M_(w)) of between 850 and 1,650 Dabased on the total weight of dextrin, dextran, isomaltooligosaccharide,and derivatives thereof in said agent, and wherein said cryoprotectanthas a weight average molecular weight (M_(w)) of between 300 and 9,500Da, such as between 300 and 7,500 Da, such as between 500 and 7,500 Da.

In one aspect, electrically charged derivatives are characterized by themolecular weight distribution of the uncharged starting materials suchas the derivatives made from the above mentioned dextrans, dextrins, andisomaltooligosaccharide. Thus in one aspect, said derivatives ofdextrans, dextrins and/or isomaltooligosaccharide have a weight averagemolecular weight (M_(w)) of between 300 and 9,500 Da, such as between300 and 7,500 Da, such as between 500 and 7,500 Da. In a further aspect,said derivatives are derivatives of isomaltooligosaccharide having aweight average molecular weight (M_(w)) of between 850 and 1,650 Da.

In one aspect, the cryoprotectant selected from the group consisting ofdextran, dextrin, and derivatives thereof has a weight average molecularweight (M_(w)) of between 300 and 9,500 Da, such as between 300 and7,500 Da, such as between 500 and 7,500 Da.

In one aspect, the cryoprotectant selected from the group consisting ofdextran, dextrin, and derivatives thereof has a weight average molecularweight (M_(w)) of between 1,650 and 7,500 Da.

In one aspect, the cryoprotectant selected from the group consisting ofdextran, dextrin, and derivatives thereof has a weight average molecularweight of between 1,650 and 7,500 Da, and a polydispersity of 1 and 5.

In one aspect, the cryoprotectant selected from the group consisting ofdextran, dextrin and derivatives thereof has a weight average molecularweight (M_(w)) of between 1,650 and 3,500 Da.

In one aspect, the cryoprotectant selected from the group consisting ofdextran, dextrin and derivatives thereof has a weight average molecularweight of between 1,650 and 3,500 Da, and a polydispersity of ≥1 and ≤5.

In one aspect, the cryoprotectant selected from the group consisting ofisomaltooligosaccharide, and derivatives thereof has a weight averagemolecular weight of between 850 and 1,650 Da.

In one aspect, the cryoprotectant selected from the group consisting ofisomaltooligosaccharide and derivatives thereof has a polydispersity of≥1 and ≤3.

In a further aspect, the cryoprotectant selected from the groupconsisting of isomaltooligosaccharide, and derivatives thereof has aweight average molecular weight (M_(w)) of between 850 and 1,150 Da.

In a further aspect, the weight fraction of isomaltooligosaccharideshaving less than 3 glucose units is less than 15% w/w. In one aspect,the weight fraction of isomaltooligosaccharides having more than 9glucose units is less than 20% w/w such as less than 15% w/w, such asless than 10% w/w. In a further aspect, the weight fraction ofisomaltooligosaccharides having less than 3 glucose units is less than15% w/w and the weight fraction of isomaltooligosaccharides having morethan 9 glucose units is less than 20% w/w such as less than 15% w/w,such as less than 10% w/w. The weight fraction may for example bedetermined as described in Preparation example 1 and 2 herein.

In one aspect, the cryoprotecting agent comprises at least 10% such as20% w/w of one or more of isomaltooligosaccharide and derivativesthereof having a weight average molecular weight (M_(w)) of between 300and 1,650 Da such as having a weight average molecular weight (M_(w)) ofbetween 850 and 1,650 Da based on the total weight of dextrin, dextran,isomaltooligosaccharide and derivatives thereof in said agent. In afurther aspect, the cryoprotecting agent comprises at least 30%, such as40%, such as 50%, such as 60%, such as 70%, such as 80%, such as 90%,such as 95% or more w/w of one or more of isomaltooligosaccharide andderivatives thereof having a weight average molecular weight (M_(w)) ofbetween 300 and 1,650 Da such as having a weight average molecularweight (M_(w)) of between 850 and 1,650 Da based on the total weight ofdextrin, dextran, isomaltooligosaccharide and derivatives thereof insaid agent.

Dextran and Derivatives Thereof

Dextran can be formed by several bacterial strains, mostlygram-positive, facultative anaerobe cocci, e.g. Leuconostoc andStreptococcus strains as for example described in “Advances in polymerscience”, Volume 205, Polysaccharides II, editor D. Klemm, SpringerVerlag. Dextrans for pharmaceutical use have typically been manufacturedby specific bacterial strains defined in the US or EuropeanPharmacopoeias such as for example by Leuconostoc Mesenteroides NCTC10817 or B512 F. The strain NCTC 10817 and B512F is publicly availablesince 1971 from National Collection Type Cultures (Central Public HealthLaboratory) UK,

Dextrans are a family of neutral branched polysaccharides consistingpredominantly of an α-(1→6) linked D-glucose having a main chain withvarying proportions of linkages and branches depending on the bacteriaused in the fermentation. The dextran molecule contains one freeterminal aldehyde group which is not shown in Formula I. The α-(1→6)linkages in dextran may vary from 50 to 97% of the total glucosidicbonds. The remaining glucosidic bonds represent α-(1→2), α-(1→3) andα-(1→4) linkages bound as branches. Formula I illustrates part of theα-(1→6)-linked glucose main chain of dextran with branching points in2-, 3- and 4-positions. Using the abovementioned strain B512F the ratioof α-(1-6) linkages ist typically 95% or above.

Extremely high values of molecular weight are found for the nativedextrans. Values ranging from 10⁷ to 4×10⁸ Daltons have been reported.In order to make the dextrans usable for many applications it istherefore necessary to hydrolyse the native dextrans to a lowermolecular weight. There are several methods known and available to theskilled person, however, the hydrolysis may be performed at approx. pH1.5, normally using hydrochloric acid, and at a temperature of approx.95° C. By the hydrolysis low molecular weight dextrans and glucose areproduced. The hydrolysate is typically purified and fractionated byvarious methods such as sedimentations with alcohol, filtrations andother various chromatographic methods including membrane filtration.

Dextrans for pharmaceutical use have typically been manufactured byspecific bacterial strains defined in the US or European Pharmacopoeiassuch as for example by Leuconostoc Mesenteroides NCTC 10817 or B512 F.The strain B512F and NCTC 10817 is as mentioned above both publiclyavailable from National Collection Type Cultures (Central Public HealthLaboratory) UK.

Among dextrans, particularly Dextran 40 and Dextran 70 have been usedfor human pharmaceutical use. Other molecular sizes such as e.g. Dextran500 and Dextran 5 and molecular weights in between are used outside thearea of cryopreservation as carriers for synthesis, for separation ofcells, as excipients in vaccines or in various other applications suchas preservation of the human cornea. Furthermore, Dextran 1 has aspecial use in humans as pre-injection of Dextran 1 exhibitshapten-inhibition and blocks human dextran antibodies, thus preventingpotential allergic reactions known to occur occasionally afteradministration of high molecular weight dextran in humans. Dextran 1,Dextran 40 and Dextran 70 are well described Pharmacopoeia products(European Pharmacopoeia 7^(th) Edition, volume 2, page 1816-1819).

Dextran is also an excellent raw material used for synthesizingwater-soluble polymers.

The following are examples of derivatives of dextran:

-   1) Hydrogenated dextran which may be synthesised by reaction of    dextran with a reducing agent such as borohydride under alkaline    conditions for example at pH 8-12 reducing aldehyde end groups into    sorbitol.-   2) Ethers of dextran which may be synthesies by methods known to the    skilled person. As an example mention can be made of    2-(Diethylamino)ethyl dextran (DEAE dextran) (shown in Reaction    scheme 1) which may be synthesised by reaction of dextran with    (2-chloroethyl)diethylammonium chloride in alkaline solution.

Another example is CarboxyC₁₋₁₀ alkyl dextran such ascarboxymethyldextran (CMD) as shown in Reaction Scheme 2 which may besynthesised by reaction with monochloric acetic acid (MCA) under strongalkaline conditions.

-   3) Esters of dextran such as dextran acetate which may be    synthesised by reaction of dextran with acetic acid anhydride.

-   4) Oxidated dextran may for example be synthesised by means of a    sodium hypochlorite in a basic aqueous solution.-   5) Partly oxidated/hydrogenated dextran. A method of preparation of    this type of derivatives is for example disclosed in U.S. Pat. No.    6,977,249 which are incorporated herein by reference. As an example    mention can be made of a dextran which is prepared by a process in    which process the molecular weight of a dextran is reduced by    hydrolysis, and functional aldehyde terminal groups thereof    converted into alcohol groups by hydrogenation; characterized in    that the hydrogenation is only partial, leaving at the most 15% by    weight reducing sugar, calculated on the total amount of carbon    hydrates, and said dextran is subsequently subjected to oxidation,    said hydrogenation and oxidation being performed to obtain dextran    having substantially all aldehyde groups converted into alcohol and    carboxylic groups, and said dextran product having no functional    aldehyde groups or functional carboxylic acid groups in the    intermediate glycosyl groups; wherein the hydrogenation is performed    by means of sodium borohydride in aqueous solution; and wherein the    oxidation is performed by means of a sodium hypochlorite in basic    aqueous solution.-   6) Further DEAE-substituted, CarboxyC₁₋₁₀ alkyl-substituted, esters    and ethers of above hydrogenated and/or oxidated dextran may be    prepared by methods known to the skilled person similar to the above    described.

Isomaltooligosaccharide and Derivatives Thereof

Isomaltooligosaccharides are glucose oligomers with an α-D-(1,6)-boundmain chain. In one aspect, the isomaltooligosaccharide described hereinis dextran-based and is made by hydrolysis of low molecular weightdextran. In a further aspect, the isomaltooligosaccharide described hasa weight average molecular weight (M_(w)) of between 300 and 1,650 Dasuch as having a weight average molecular weight (M_(w)) of between 850and 1,650 Da. In one aspect, the isomaltooligosaccharide describedherein is hydrolysed dextran having a weight average molecular weight(M_(w)) of between 850 and 1,650 Da.

Starting from isomaltooligosaccharide derivatives thereof characterisedby the changing of the reducing aldehyde end groups intoglycitol/sorbitol may be prepared. The conversion fromisomaltooligosaccharide to hydrogenated isomaltooligosaccharide may beperformed by treating the isomaltooligosaccharide with a reducing agent,such as e.g. borohydride under alkaline conditions as shown in belowReaction scheme 4:

With the processes described above for making derivatives of dextran,derivatives of isomaltooligosaccharide may also be made for example bythe reaction with (2-chloroethyl)diethylammonium chloride making2-(diethylaminodextran)ethyl (DEAE) isomaltooligosaccharide. Anotheroption is to make isomaltooligosaccharide derivatives by reaction withmonochloroacetic acid (MCA) for synthesising carboxymethylisomaltooligosaccharide. To the skilled person it will be obvious tomake further derivatives of hydrogenated isomaltooligosaccharide byreaction with respectively (2-chloroethyl)diethylammonium chloride andmonochloroacetic acid as described above under dextran andisomaltooligosaccharide.

Oligo-Isomaltose

With dextrans as starting materials and Dextran 1(isomaltooligosaccharide) as intermediary, it is possible to synthesiseoligo-isomaltose, characterized by a complete lack of branchingside-chains α-(1→2), α-(1→3) and α-(1→4) defining the dextran molecules.In the present context oligoisomaltose is considered as a subset ofisomaltooligosaccharide. Thus, in one aspect, oligoisomaltose has aweight average molecular weight (Mw) of between 300 and 1,650 Da such asbetween 850 and 1,650 Da, preferably between 850 and 1,150 Da, andhaving a complete lack of branching side-chains α-(1→2), α-(1→3) andα-(1→4). Obviously the same synthesis of derivatives described above forisomaltooligosaccharide can be performed using oligo-isomaltose.

Dextrin and Derivatives Thereof

Dextrins are a group of low-molecular-weight carbohydrates produced bythe hydrolysis of starch. Dextrins are as dextrans polymers consistingof a mixture of molecules with varying length of the backbone glucosechain. Before using the dextrins they are typically purified andfractionated e.g. by applying hydrolysis or one or more alcoholsedimentation processes and/or by various chromatographic methodsincluding membrane processing applying one or more membranes withspecific cut-off values in order to obtain the desired molecular seizeand weight distribution.

Dextrins are mixtures of polymers of D-glucose units linked by α-(1→4)or α-(1→6) glycosidic bonds as shown in Formula II.

Derivatives of dextrin may be made by methods known to the skilledperson similar to the above described under dextran andisomaltooligosaccharide.

Cryopreservation

In order to avoid contamination of the sample to be cryopreserved it ispreferred that the cryoprotectant is sterile, and that other optionalcomponents of the cryopreservation agent/composition also are sterile.

In some applications it might be useful to supplement a cryoprotectantselected from the group consisting of dextrin, dextran,isomaltooligosaccharide, and derivatives thereof with an additionalcryoprotectant in order to reduce the concentration of such additionalcryoprotectant, preferably to non-toxic concentrations. This may beparticular useful for specific cell types, like hepatocytes orpluripotent stem cells. In a further aspect, the cryoprotecting agentthus further comprises at least one additional cryoprotectant selectedfrom the group consisting of acetamide, agarose, alginate, 1-analine,albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform,choline, diethylene glycol, dimethyl acetamide, dimethyl formamide,dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol,formamide, glucose, glycerol, a-glycerophosphate, glycerol monoacetate,glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride,magnesium sulfate, maltose, mannitol, mannose, methanol, methylacetamide, methylformamide, methyl ureas, phenol, pluronic polyols,polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol,pyridine N-oxide, ribose, serine, sodium bromide, sodium chloride,sodium iodide, sodium nitrate, sodium sulfate, sorbitol, sucrose,trehalose, triethylene glycol, trimethylamine acetate, urea, valine andxylose. In one aspect, said additional cryoprotectant is DMSO. Anadvantage of adding DMSO in a reduced amount may be that for veryfragile cells an additional protection may be obtained. In a preferredaspect, the cryoprotecting agent is free of or substantially free ofDMSO. Thus, in yet a preferred aspect, said dextrin, dextran,isomaltooligosaccharide, or a derivative thereof is the onlycryoprotectant in the cryoprotecting agent. A cryoprotecting agent freeor substantially free of DMSO may not require washing after thawing ofthe sample. The thawed sample may then be directly suspended in aculture medium to immediately start a culturing process without havingto wash the sample or may be directly used in a patient without awashing step that potentially leads to substantial cell loss. Anotheradvantage using a cryoprotecting agent free of or substantially free ofDMSO is that the sample may be exposed to the cryoprotectant for alonger period without damage, enabling a more efficient working process.

In another aspect, the cryoprotecting agent further comprises at leastone anti-freeze protein and/or anti-freeze glycoprotein such as in anamount of from 0.01 to 1 mg/mL of the cryoprotecting agent. An exampleof an anti-freeze glycoprotein is Type I AFP from longhorn sculpin,which is a single, long amphipathic alpha helix.

The cryoprotecting agent or composition may comprise further substancesfor improving the viability of the sample. As examples of suchsubstances mention can be made of IAPs (inhibitors of apoptosis),inhibitors of the rho-associated protein kinase (ROCK) signalingpathways, growth factors such as EGF, FGF, PDGF, IGF, EPO, BDNF, TGF,TNF, VEGF. In a further aspect, mention can be made of any serumcomponents of human, bovine, equine, canine origin. The cryoprotectingagent or composition may also comprise a growth medium. In one aspect, agrowth medium comprising β-catenin/P300 antagonist and an Activin/TGFβligand, such as for example ID-8 in conjunction with Activin and TGFβ,may be used. This type of medium is especially useful for culturing ofpluripotent stem cells, in particular embryonic stem cells as forexample described in WO 2013/054112. Another example is the standardknock-out medium comprising KnockOut Serum Replacement, DMEM/F12 withGlutaMAX™ supplement, FGF, NEAA and BME. Another example is the mTSER™system. Other examples of growth media depending on the sample to becryopreserved is well known to the skilled person.

The cryoprotecting agent as disclosed herein may be in the form of apowder such as a lyophilized or spray dried powder.

In a further aspect, said cryoprotecting agent is in the form of asolution. The agent may thus further comprise a solvent such as forexample sterile water. In one aspect, said agent comprises from 30% to70% w/w of said cryoprotectant, such as from 40% to 65% w/w or from 50%to 60% w/w of said cryoprotectant.

The sample such as cells, tissue or organs to be cryopreserved can alsobe in contact with a freezing compatible pH buffer comprised mosttypically of at least a basic salt solution, an energy source (forexample, glucose) and a buffer capable of maintaining a neutral pH atlow temperatures. Well known materials include, for example, Dulbecco'sModified Eagle Medium (DMEM). This material may also be included as partof the cryopreservation composition and/or agent.

One aspect disclosed herein is a cryopreservation composition comprisinga cryoprotecting agent as described herein, which cryopreservationcomposition further comprises a sample to be cryopreserved.

A further aspect disclosed herein is a cryopreserved compositioncomprising a cryoprotecting agent and a sample which has beencryopreserved or is in the process of being cryopreserved. As describedherein the term, “a cryopreserved composition” means either “acryopreservation composition” which is in the process of beingcryopreserved or already has been cryopreserved.

A further aspect disclosed herein is a cryopreserved compositioncomprising a growth medium or substrate for the sample to becryopreserved.

In one aspect, the sample is selected from the group consisting oforgans, cells, and tissues such as mammalian. In a further aspect, thesample is organs, cells, blood or tissues. Examples of such cells to becryopreserved are in-vitro-cultured cells including primary cells, celllines, in vitro-sorted cells including human blood cells, and fertilizedeggs of animal and human origin. Further examples are sperm cells,embryonic stem cells, IPS cells, mesenchymal stem cells, haemopoieticstem cells, neuronal stem cells, umbilical cord blood stem cells,hepatocytes, nerve cells, cardiomyocytes, vascular endothelial cells,vascular smooth muscle cells and blood cells. In a further aspect, thesample is cells selected from the group consisting of mesencymal stemcells, hematopoietic stem cells, embryonic stem cells, IPS cells,keratinocytes, preferable hematopoietic stem cells such as CD34 positiveblood stem cells, mesenchymal stem cells embryonic stem cells and IPScells. In a further aspect the sample is selected from the groupconsisting of mesenchymal stem cells and hematopoietic stem cells. Inone aspect, the cell is of animal or human origin. Examples of organsare lung, liver, kidney, heart, ovaries and pancreas. Examples oftissues are tissues of bone marrow, skin, ovaries, testis, bloodvessels, connective tissue, preferable tissues of ovaries and connectivetissue. In a further aspect, the blood is selected from the groupconsisting of umbilical cord blood, and mobilized peripheral blood,preferable umbilical cord blood. In a further aspect, the sample is acell-containing body fluid such as blood, menstrual fluid or amnioticfluid.

Depending on the specific sample to be cryoprotected, the cryoprotectantis typically present in the composition to be cryopreserved in an amountfrom 1 to 50% w/w such as from 2 to 50% w/w, such as from 4 to 45% w/w,or from 6 to 20% w/w, or from 6 to 12% w/w, or preferably from 6 to 10%w/w, or more preferably from 7 to 9% w/w. In one aspect, thecryoprotectant is present in the composition to be cryopreserved in anamount of at the most 60% w/w, such as in an amount of at the most 55%w/w, such as in an amount of at the most 50% w/w, such as in an amountof at the most 45% w/w, such as in an amount of at the most 40% w/w,such as in an amount of at the most 35% w/w. In another aspect, thecryoprotectant is typically present in an amount of at the least 2% w/w,such as present in an amount of at the least 4% w/w, such as present inan amount of at the least 6% w/w, such as present in an amount of at theleast 6% w/w, such as present in an amount of at the least 7% w/w.

If the composition to be cryoprotected comprises an additionalcryoprotectant such as DMSO the additional cryoprotectant typically ispresent in an amount of less than 8% w/w, such as from 1-8% w/w, such asfor example in an amount below 5% w/w, such as below 4% w/w such as from1-4% w/w.

In conventional cryopreservation techniques, a sample is harvested,suspended in a storage solution, and then preserved by freezing. Whenthe sample such as cells are to be used, they are thawed, for example,cells taken from human donor sources are brought back to the normalhuman body temperature (i.e., approximately 37° C.), and then placed ina cell culture medium.

In the present method of cryopreservation, the sample is protectedduring cryopreservation by being brought into contact with acryoprotecting agent as described herein prior to freezing to thecryopreservation temperature. By being brought into contact with thecryoprotecting agent is meant that the sample is made to be in contactin some manner with the cryoprotectant so that during the reduction oftemperature to the cryopreservation temperature, the sample is protectedby the cryoprotectant in the cryopreservation composition. For example,the cells may be brought into contact with the cryoprotecting agent byfilling the appropriate wells of a plate to which the cells to beprotected are attached, by suspending the cells in a solution of thecryoprotecting agent or by adding the cryoprotecting agent for examplein freeze dried form to the cells, blood or organ already in a solutionof for example buffer, or by resuspending the cell pellet aftercentrifugation in the cryoprotecting agent bringing the cells into asolution etc.

In one aspect, disclosed herein is a method of cryopreserving a sample,comprising the steps of bringing a sample to be cryopreserved intocontact with a cryoprotecting agent as defined herein to obtain acryopreservation composition and subsequently reducing the temperatureof the cryopreservation composition to a cryopreservation temperature.

In a further aspect, a method of cryopreserving a composition as definedherein by reducing the temperature of said composition to acryopreservation temperature is disclosed herein.

The rate of change from room temperature to 1-2° C. below the freezingpoint of the solution may have a major effect on ultimate viability ifthe cells are sensitive to thermal shock.

Between 3.5° C. and −5° C., the sample is normally induced to freezeeither by the introduction of an ice crystal, by touching the surface ofthe media with a cold probe, by mechanical vibration, or by rapidlylowering the temperature until ice nucleation occurs. Since freezing isan exothermic process, heat must be conducted away from the freezingsolution. This may be done either by keeping the samples immersed in aliquid with a low freezing point or by providing a substantial heatsink. As ice forms in the extracellular media, more and more free waterbecomes bound in the ice phase. Cell membranes, being hydrophobic, actas a barrier for the nucleation of intracellular ice and thereforeunfrozen cells are exposed to an increasingly hypertonic solution. Theextracellular salt concentration increases as a consequence of watersequestration into ice. The unfrozen cells shrink due to the transportof water out of the cell in response to the osmotic imbalance betweenthe intracellular and extracellular fluid phases. The sample is thencooled at a finite rate which must be optimized for each cell type.

The optimal rate of cooling is determined by the permeability of thecell membrane to water, the surface-to-volume ratio of the cell, alongthe type and concentration of cryoprotective additives in thecryoprotecting agent as described herein. For most nucleated mammaliancells frozen in glycerol or DMSO, the optimal cooling rate usually isbetween about 0.3 to 10° C. per minute. Continuous cooling between about4° C. and −80° C. is the most commonly used. Once the sample reachesapproximately −80° C., it can be transferred directly into liquidnitrogen (−196° C.) or into the vapor phase of liquid nitrogen forstorage. Another method used for cryopreservation is the vitrificationtechnology in which it is possible to obtain very fast cooling rates of1000° C.-2000° C./min. With this technology a specialized vitrificationdevice, containing the cryopreservation composition with the sample, isdirectly placed into liquid nitrogen. In one aspect, thecryopreservation temperature is reached at a rate of 0.05-15, such as0.1-10, such as 0.2-8, such as 0.3-6, such as 0.4-4, such as 0.5-2° C.per minute. In another aspect the cryopreservation temperature isreached at a rate of 500-3000, such as 800-2500, such as 1000-2000, suchas 1200-1800° C. per minute.

The duration of viable cell storage at liquid nitrogen temperature isdependent predominantly on the rate of generation of free radicalscaused by the cosmic ray background.

For example, the half-life for mammalian embryos stored in liquidnitrogen has been estimated to be approximately 30,000 years. It isimportant not to allow frozen cells to warm above their storagetemperature for even brief periods of time. Intermittent warmingpromotes rapid migratory recrystallization, which can damage cellularstructure and decrease overall viability.

In yet a further aspect, the sample are thawed after cryopreservation.The optimal rate of thawing of the sample is dependent on the freezingconditions used and the specific sample to be preserved. In general, forsingle cells frozen in suspension, and for tissues such as heart valves,a rapid rate of warming is desirable. Such rapid warming limits thegrowth of ice crystals in the frozen samples and is often an absoluterequirement for high survival. With many tissues this warming can beaccomplished by agitating the sample in a 37-42° C. water bath. Therationale for rapid warming is that it limits the growth of ice crystalswhich were formed during cooling.

Some tissues may be sensitive to rapid warming. This is due to transientosmotic shock, because the cells are exposed to an extracellularhypertonic solution as the ice melts and are forced to rehydrate inorder to maintain their osmotic equilibrium. For other, more sensitive,samples, metabolic processes can be reactivated or brought up to normallevels by successive dilutions using serum or other high molecularweight polymers in the thawing medium.

Upon completion of the thawing procedure, the cells are still exposed tomultimolar concentrations of cryoprotecting agents which must begradually diluted to return the cells to an isotonic media. Formammalian cells, a stepwise dilution protocol is typically used. Thedilution of the sample is normally carried out at preferably 37° C., soas to reduce the effects of both osmotic shock and cryoprotectanttoxicity. In a further aspect, the concentration of said cryoprotectantis from 4 to 45% w/w, such as from 4 to 20% w/w, such as from 5 to 15%w/w, or from 6 to 12% w/w, or preferably from 6 to 10% w/w, or morepreferably from 7 to 9% w/w cryoprotectant.

In a further aspect, the temperature of the sample in thecryopreservation composition is reduced to a cryopreservationtemperature below −50° C., such as between −50° C. to −196° C., such asbetween −80° C. to −196° C.

In one aspect, the cryoprotecting agent is used in a banking method. Inone aspect, the cryoprotecting agent is used in a clinical bankingmethod. In one aspect, the cryoprotecting agent is used in a mobilizedperipheral blood banking method.

In one aspect, the cryoprotecting agent is used in a clinical bankingmethod such as in stem cell transplantation for malignant diseases or inan organ transplantation. In one aspect, the cryoprotecting agent isused in a mobilized peripheral blood banking method, marrow bankingmethod or in an umbilical cord banking method.

In one aspect, the cryoprotecting agent is used in a marrow bankingmethod or in an umbilical cord banking method. In one aspect, thecryoprotecting agent is used in a fat tissue banking method or in adental pulp tissue banking method. In a further aspect, thecryoprotecting agent is used in a reproduction banking method.

In the Following Further Embodiments are Disclosed

1. A cryoprotecting agent comprising a cryoprotectant being one or moreof: dextrin, dextran, isomaltooligosaccharide, and derivatives thereof,such as being one or more of: dextran, isomaltooligosaccharide, andderivatives thereof, and

-   -   a) wherein said cryoprotecting agent comprises at least 1% w/w        of one or more of isomaltooligosaccharide and derivatives        thereof based on the total weight of dextrin, dextran,        isomaltooligosaccharide and derivatives thereof in said agent,        and/or    -   b) wherein said cryoprotectant has a weight average molecular        weight (M_(w)) of between 300 and 9,500 Da.        2. A cryoprotecting agent comprising one or more cryoprotectants        selected from the group consisting of dextrin, dextran,        isomaltooligosaccharide, and derivatives thereof, such as        selected from the group consisting of dextran,        isomaltooligosaccharide, and derivatives thereof, and    -   a) wherein said cryoprotecting agent comprises at least 1% w/w        of one or more of isomaltooligosaccharide and derivatives        thereof having a weight average molecular weight (M_(w)) of        between 300 and 1,650 Da based on the total weight of dextrin,        dextran, isomaltooligosaccharide and derivatives thereof in said        cryoprotecting agent, or    -   b) wherein said cryoprotectant has a weight average molecular        weight (M_(w)) of between 300 and 9,500 Da, or    -   c) wherein said cryoprotecting agent comprises at least 1% w/w        of one or more of isomaltooligosaccharide and derivatives        thereof having a weight average molecular weight (M_(w)) of        between 300 and 1,650 Da based on the total weight of dextrin,        dextran, isomaltooligosaccharide and derivatives thereof in said        cryoprotecting agent, and said cryoprotectant has a weight        average molecular weight (M_(w)) of between 300 and 9,500 Da.        3. The agent according to any one of embodiments 1-2, wherein        said cryoprotectant has a weight average molecular weight        (M_(w)) of between 300 and 7,500 Da.        4. The agent according to any one of embodiments 1-3, wherein        said cryoprotectant is one or more selected from the group        consisting of isomaltooligosaccharide, and derivatives thereof        having a weight average molecular weight (M_(w)) of between 300        and 1,650.        5. The agent according to any one of embodiments 1-4, wherein        said cryoprotecting agent comprises at least 1% w/w of one or        more of isomaltooligosaccharide and derivatives thereof having a        weight average molecular weight (M_(w)) of between 300 and 1,650        Da based on the total weight of dextrin, dextran,        isomaltooligosaccharide and derivatives thereof in said        cryoprotecting agent.        6. The agent according to any one of embodiments 1-5, wherein        said isomaltooligosaccharide, and derivatives has a weight        average molecular weight (M_(w)) of between 850 and 1,650 Da.        7. The agent according to any one of embodiments 1-6 for        cryopreserving a sample, and wherein said sample is selected        from the group consisting of organs, cells, and tissue such as        selected from the group consisting of mammalian organs,        mammalian cells, and mammalian tissues.        8. The agent according to any one of embodiments 1-7 for        cryopreserving a sample, and wherein said sample is for        transplantation.        9. The agent according to any one of embodiments 1-8 for        cryopreserving a sample, and wherein said sample is functional        after cryopreservation.        10. The agent to any one of embodiments 1-9, wherein said sample        is an organ, which organ is functional as measured by        physiological function of said organ after cryopreservation,        and/or wherein said sample is tissue, which tissue is functional        as measured by ability of such tissue to integrate with        surrounding tissue and/or wherein said sample is cells, which        cells are functional as measured by viability of said cells        after cryopreservation.        11. The agent according to any one of embodiments 1-10, wherein        said cryoprotectant has a weight average molecular weight        (M_(w)) of between 1,650 and 7,500 Da.        12. The agent according to any one of embodiments 1-11, wherein        said cryoprotectant has a weight average molecular weight        (M_(w)) of between 500 and 3,500 Da.        13. The agent according to any one of embodiments 1-12, wherein        said cryoprotectant has a weight average molecular weight        (M_(w)) of between 1,650 and 3,500 Da.        14. The agent according to any one of embodiments 1-13, wherein        said cryoprotectant has a polydispersity Pd, wherein Pd is ≥1        and ≤5.        15. The agent according to any one of embodiments 1-14, wherein        said cryoprotectant has a weight average molecular weight        (M_(w)) of between 850 and 1,650 Da.        16. The agent according to any one of embodiments 1-15, wherein        said cryoprotectant has a weight average molecular weight        (M_(w)) of between 850 and 1,150 Da.        17. The agent according to any one of embodiments 1-16, wherein        said cryoprotectant has a polydispersity Pd, wherein Pd is ≥1        and ≤3.        18. The agent according to any one of embodiments 1-17        comprising at least 10% w/w of one or more of        isomaltooligosaccharide and derivatives thereof based on the        total weight of dextrin, dextran, isomaltooligosaccharide and        derivatives thereof in said agent.        19. The agent according to any one of embodiments 1-18, wherein        the weight fraction of isomaltooligosaccharides having less than        3 glucose units is less than 15% w/w.        20. The agent according to any one of embodiments 1-19, wherein        the weight fraction of isomaltooligosaccharides having more than        9 glucose units is less than 20% w/w.        21. The agent according to any one of embodiments 1-20, wherein        the weight fraction of isomaltooligosaccharides having less than        3 glucose units is less than 15% and the weight fraction of        isomaltooligosaccharides having more than 9 glucose units is        less than 20% w/w such as less than 15% w/w, such as less than        10% w/w.        22. The agent according to any one of embodiments 1-21        comprising at least 30% w/w, at least 40% w/w, at least 50% w/w,        at least 60% w/w, at least 70% w/w, at least 80% w/w, at least        90% w/w, at least 95% w/w or more of one or more of        isomaltooligosaccharide and derivatives thereof based on the        total weight of dextrin, dextran, isomaltooligosaccharide and        derivatives thereof in said agent.        23. The agent according to any one of embodiments 1-22, wherein        said cryoprotectant is isomaltooligosaccharide, such as        isomaltooligosaccharide having a weight average molecular weight        (M_(w)) of between 850 and 1,150 Da.        24. The agent according to any one of embodiments 1-23, wherein        said isomaltooligosaccharide and derivatives thereof is        dextran-based.        25. The agent according to any one of embodiments 1-24, wherein        said derivative is a derivative of dextrin, dextran, and        isomaltooligosaccharide selected from the group consisting of        hydrogenated isomaltooligosaccharide, hydrogenated dextran,        hydrogenated dextrin, oxidised isomaltooligosaccharide, oxidised        dextran, oxidised dextrin, ester of dextrin, ester of dextran,        ester of isomaltooligosaccharide, ether of dextrin, ether of        dextran, ether of isomaltooligosaccharide, partly        hydrogenated/oxidised dextrin, partly hydrogenated/oxidised        dextran and partly hydrogenated/oxidised        isomaltooligosaccharide, and derivatives thereof such as        selected from the group consisting of hydrogenated        isomaltooligosaccharide, hydrogenated dextran, hydrogenated        dextrin, oxidised isomaltooligosaccharide, oxidised dextran,        oxidised dextrin, DEAE-dextrin, DEAE-dextran,        DEAE-isomaltooligosaccharides, carboxyC₁₋₁₀alkyl-dextrin,        carboxyC₁₋₁₀alkyl-dextran,        carboxyC₁₋₁₀alkyl-isomaltooligosaccharide, ester of dextrin,        ester of dextran and ester of isomaltooligosaccharide, and        derivatives thereof.        26. The agent according to any one of embodiments 1-25, wherein        said derivative is a hydrogenated isomaltooligosaccharide, an        oxidised isomaltooligosaccharide, DEAE-isomaltooligosaccharid, a        carboxyC₁₋₁₀alkyl oxidised isomaltooligosaccharide or an        carboxyC₁₋₁₀alkyl isomaltooligosaccharide.        27. The agent according to any one of embodiments 1-26, wherein        said derivative is hydrogenated isomaltooligosaccharide, such as        hydrogenated isomaltooligosaccharide having a weight average        molecular weight (M_(w)) of between 850 and 1,150 Da.        28. The agent according to any one of embodiments 25-27, wherein        said carboxyC₁₋₁₀alkyl isomaltooligosaccharide is carboxymethyl        isomaltooligosaccharide or carboxyethyl isomaltooligosaccharide.        29. The agent according to any one of embodiments 1-28,        comprising at least one additional cryoprotectant selected from        the group consisting of acetamide, agarose, alginate, 1-analine,        albumin, ammonium acetate, butanediol, chondroitin sulfate,        chloroform, choline, diethylene glycol, dimethyl acetamide,        dimethyl formamide, dimethyl sulfoxide (DMSO), erythritol,        ethanol, ethylene glycol, formamide, glucose, glycerol,        a-glycerophosphate, glycerol monoacetate, glycine, hydroxyethyl        starch, inositol, lactose, magnesium chloride, magnesium        sulfate, maltose, mannitol, mannose, methanol, methyl acetamide,        methylformamide, methyl ureas, phenol, pluronic polyols,        polyethylene glycol, polyvinylpyrrolidone, proline, propylene        glycol, pyridine N-oxide, ribose, serine, sodium bromide, sodium        chloride, sodium iodide, sodium nitrate, sodium sulfate,        sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine        acetate, urea, valine and xylosen.        30. The agent according to any one of embodiments 1-29, wherein        said additional cryoprotectant is DMSO.        31. The agent according to any one of embodiments 1-30 which is        substantially free of DMSO.        32. The agent according to embodiment 31 which is free of DMSO.        33. The agent according to any one of embodiments 1-32        comprising a cryoprotectant as defined in embodiment 1 as the        only cryoprotectant.        34. The agent according to any one of embodiments 1-33, wherein        said agent is in the form of a powder.        35. The agent according to any one of embodiments 1-34, wherein        said agent is in the form of a lyophilized or spray dried        powder.        36. The agent according to any one of embodiments 1-35, wherein        said agent is in the form of a solution.        37. The agent according to any one of embodiments 1-36, wherein        said agent comprises from 30% to 70% w/w of said cryoprotectant,        such as from 40% to 65% w/w or from 50% to 60% w/w of said        cryoprotectant.        38. The agent according to any one of embodiments 1-37, wherein        said agent comprises from 30% to 70% w/w of said additional        cryoprotectant, such as from 40% to 65% w/w or from 50% to 60%        w/w of said additional cryoprotectant.        39. The agent according to any one of embodiments 1-38 further        comprising a growth medium or substrate for a sample to be        cryopreserved.        40. The agent according to any one of embodiments 1-39 further        comprising any proteins belonging to the IAPs (Inhibitors of        apoptosis), inhibitors of the rho-associated protein kinase        (ROCK) signaling pathways, and/or any growth factor such as EGF,        FGF, PDGF, IGF, EPO, BDNF, TGF, TNF and/or VEGF.        41. The agent according to any one of embodiments 1-40 further        comprising any serum component of human, bovine, equine, or        canine origin.        42. The agent according to any one of embodiments 1-41, wherein        said cryoprotectant is sterile.        43. A cryopreservation composition comprising a cryoprotecting        agent as defined in any one of embodiments 1-42, which        cryopreservation composition further comprises a sample to be        cryopreserved.        44. The cryopreservation composition according embodiment 43,        wherein the sample is selected from the group consisting of        organs, cells such as isolated cells or cell-containing bodily        fluids for example blood, and tissues.        45. The cryopreservation composition according embodiment 44,        wherein the sample is selected from the group consisting of        mammalian organs, mammalian cells, and mammalian tissues, such        as a sample selected from the group consisting of mammalian        organs, mammalian cells, and mammalian tissues for        transplantation.        46. The cryopreservation composition according to any one of        embodiments 43-45, wherein said sample is cells selected from        the group consisting of somatic cells, including all kind of        tissue derived cells such as mesenchymal stem cells,        tissue-specific progenitor cells, keratinocytes, fibroblasts,        chondrocytes, bone cells, or cardiomyocytes, blood derived cells        such as hematopoietic stem cells, macrophages, plates,        erythrocytes, or stem cells, including all types of pluripotent        cells, totipotent cells and unipotent cells, and germ layer        cells.        47. The cryopreservation composition according to any one of        embodiments 43-46, wherein said sample is cells selected from        the group consisting of keratinocytes, fibroblasts, mesenchymal        stem cells, macrophages, and hematopoietic stem cells such as        CD34 positive blood stem cells.        48. The cryopreservation composition according to any one of        embodiments 43-45, wherein said sample is tissue selected from        the group consisting of ovarian tissue, testicular tissue,        umbilical cord tissue, placental tissue, connective tissue,        cardiac tissue, tissue from muscle, bone, and cartilage tissue,        endocrine tissue, and neural tissue.        49. The cryopreservation composition according to any one of        embodiments 43-45, wherein said sample is a cell-containing        bodily fluid selected from the group consisting of blood such as        umbilical cord blood, peripheral blood, and mobilized peripheral        blood, amniotic fluid, semen, cerebrospinal fluid, menstrual        fluid blood, and bone marrow aspirates.        50. The cryopreservation composition according to any one of        embodiments 43-45, wherein said sample is an organ selected from        the group consisting of lung, heart, kidney, liver, umbilical        cord and ovaries.        51. The cryopreservation composition according to any one of        embodiments 43-45 comprising said cryoprotectant in an amount        from 1 to 50% w/w such as from 2 to 50% w/w, or from 4 to 45%        w/w, or from 6 to 12% w/w, or preferably from 6 to 10% w/w, or        more preferably from 7 to 9% w/w.        52. The cryopreservation composition according to any one of        embodiments 43-51, wherein said composition comprises DMSO in an        amount of less than 8% w/w, such as from 1-8%.        53. The cryopreservation composition according to any one of        embodiments 43-52, wherein said sample is functional after        cryopreservation.        54. The cryopreservation composition according to any one of        embodiments 43-53, wherein said composition comprises DMSO in an        amount of less than 8% w/w, less than 4% w/w such as from 1-4%.        55. A method of cryopreserving a sample, comprising bringing a        sample to be cryopreserved into contact with a cryoprotecting        agent as defined in any one of embodiments 1-42 to obtain a        cryopreservation composition and subsequently reducing the        temperature of the cryopreservation composition to a        cryopreservation temperature.        56. The method according to embodiment 55, wherein the        cryopreservation composition is as defined in any one of        embodiments 43-54.        57. A method of cryopreserving a composition as defined in any        one of embodiments 43-56 by reducing the temperature of said        composition to a cryopreservation temperature.        58. The method according to any one of embodiments 55-57,        wherein the cryopreservation temperature is reached at a rate of        0.05-15, such as 0.1-10, such as 0.2-8, such as 0.3-6, such as        0.4-4, such as 0.5-2° C. per minute.        59. The method according to any one of embodiments 55-58,        wherein the concentration of said cryoprotectant is from 4 to        20% w/w such as from 5 to 15% w/w, or from 6 to 12% w/w, or        preferably from 6 to 10% w/w, or more preferably from 7 to 9%        w/w.        60. The method according to any one of embodiments 55-59,        wherein the temperature of the sample in the cryopreservation        composition is reduced to a temperature below −50° C., such as        between −50° C. to −196° C., such as −80° C. to −196° C.        61. The method according to any one of embodiments 55-60,        wherein the sample is thawed after cryopreservation.        62. The method according to any one of embodiments 55-61,        wherein said sample is functional after cryopreservation.        63. The method according to any one of embodiments 55-62,        wherein the sample to be cryopreserved is selected from the        group consisting of organs, cells, and tissues.        64. The method according to any one of embodiments 55-63, which        is in a clinical banking method.        65. The method according to any one of embodiments 55-64, which        is in a banking method such as a mobilized peripheral blood        banking method, marrow banking method, a fat tissue banking        method, a dental pulp tissue banking method, a reproduction        banking method or in an umbilical cord banking method.        66. Use of a cryoprotecting agent comprising one or more        cryoprotectants selected from the group consisting of dextrin,        dextran, isomaltooligosaccharide, and derivatives thereof, such        as selected from the group consisting of dextran,        isomaltooligosaccharide, and derivatives thereof; and a) wherein        said cryoprotecting agent comprises at least 1% w/w of one or        more of isomaltooligosaccharide and derivatives thereof having a        weight average molecular weight (M_(w)) of between 300 and 1,650        Da based on the total weight of dextrin, dextran,        isomaltooligosaccharide and derivatives thereof in said        cryoprotecting agent, or b) wherein said cryoprotectant has a        weight average molecular weight (M_(w)) of between 300 and 9,500        Da, or c) wherein said cryoprotecting agent comprises at least        1% w/w of one or more of isomaltooligosaccharide and derivatives        thereof having a weight average molecular weight (M_(w)) of        between 300 and 1,650 Da based on the total weight of dextrin,        dextran, isomaltooligosaccharide and derivatives thereof in said        cryoprotecting agent, and said cryoprotectant has a weight        average molecular weight (M_(w)) of between 300 and 9,500 Da,        for cryopreserving a sample, and wherein said sample is selected        from the group consisting of organs, cells, and tissue.        67. The use of a cryoprotecting agent according to embodiment        66, wherein said cryoprotectant has a weight average molecular        weight (M_(w)) of between 300 and 7,500 Da.        68. Use of a cryoprotecting agent selected from the group        consisting of isomaltooligosaccharide and derivatives thereof        having a weight average molecular weight (M_(w)) of between 300        and 1,650 Da such as having a weight average molecular weight        (M_(w)) of between 850 and 1,650 Da, for cryopreserving a sample        wherein said sample is selected from the group consisting of        organs, cells, and tissue.        69. The use of a cryoprotecting agent as defined in any one of        embodiments 1-42 for cryopreserving a sample such as a sample        selected from the group consisting of organs, cells, and tissue.        70. The use according to any one of embodiments 66-69 comprising        bringing said sample to be cryopreserved into contact with said        agent to obtain a cryopreservation composition and subsequently        reducing the temperature of the cryopreservation composition to        a cryopreservation temperature.        71. Use of a cryopreservation composition as defined in any one        of embodiments 43-54 for cryopreserving a sample by reducing the        temperature of said composition to a cryopreservation        temperature.        72. The use according to any one of embodiments 66-71, wherein        the cryopreservation temperature is reached at a rate of        0.05-15, such as 0.1-10, such as 0.2-8, such as 0.3-6, such as        0.4-4, such as 0.5-2° C. per minute.        73. The use according to any one of embodiments 66-72, wherein        the concentration of said cryoprotectant is from 4 to 20% w/w        such as from 5 to 15% w/w, or from 6 to 12% w/w, or preferably        from 6 to 10% w/w, or more preferably from 7 to 9% w/w        cryoprotectant.        74. The use according to any one of embodiments 66-73, wherein        the temperature of the sample in the cryopreservation        composition is reduced to a temperature below −50° C., such as        between −50° C. to −196° C., such as −80° C. to −196° C.        75. The use according to any one of embodiments 66-74, wherein        the sample is thawed after cryopreservation.        76. The use according to any one of embodiments 66-75, wherein        said sample is functional after cryopreservation,        77. The use according to any one of embodiments 66-76, wherein        the sample to be cryopreserved is selected from the group        consisting of organs, cells, and tissues for transplantation.        78. The use according to any one of embodiments 66-77, which is        in a banking method such as a mobilized peripheral blood banking        method, marrow banking method, fat tissue banking method, a        dental pulp tissue banking method, a reproduction banking method        or in an umbilical cord banking method.        79. The use according to any one of embodiments 66-78, which is        in a clinical banking method.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed compositions, methods and system of the present invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the present invention. Although the present invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments.

Preparation Example 1 Production of Isomaltooligosaccharide 1

Hydrolysis of Low Molecular Dextran

3345 kg hydrolysed dextran collected as permeate from a membrane havinga cut-off value <5,000 Daltons, is hydrolysed at pH 1.5 at a temperatureof 95° C.

The hydrolysis is monitored chromatographically using gel permeationchromatography (GPC), and is terminated by cooling when the molecularweight of the material being hydrolysed is estimated to have achievedthe desired value, i.e. a weight average molecular weight of 850-1,150Daltons.

By the hydrolysis low molecular weight isomaltooligosaccharide isproduced but also glucose is formed. After cooling and neutralizationthe amount of glucose and very low molecular weight oligomers is reducedby membrane processes having a cut-off value of 340-800 Daltons. Afterthis process, the content of isomaltooligosaccharide is determined byoptical rotation (α_(D)20˜200) to be 915 kg, and the amount of reducingsugar is determined by use of Somogyi's reagent to be 22.5%.

TABLE 1 Results from GPC analysis. Number of glucose units In RelativeContribution Relative Contribution molecule AUPW AUPW Mi to M_(W) AUPNAUPN to M_(n) 1 0.48 0.0028 180 1 0.0027 0.0130 2 2 5.94 0.0350 342 120.0174 0.0846 29 3 21.76 0.1282 504 65 0.0432 0.2103 106 4 28.3 0.1667666 111 0.0425 0.2070 138 5 26.94 0.1587 828 131 0.0325 0.1585 131 622.58 0.1330 990 132 0.0228 0.1111 110 7 17.43 0.1027 1152 118 0.01510.0737 85 8 13.46 0.0793 1314 104 0.0102 0.0499 66 9 9.73 0.0573 1476 850.0066 0.0321 47 10 6.91 0.0407 1638 67 0.0042 0.0206 34 11 4.89 0.02881800 52 0.0027 0.0132 24 12 3.66 0.0216 1962 42 0.0019 0.0091 18 13 2.50.0147 2124 31 0.0012 0.0057 12 14 5.2 0.0306 2286 70 0.0023 0.0111 25169.78 1.0000 1020 0.2053 1.0000 827 AUPW: Area under peak (M_(w)) AUPN:Area under peak (M_(n))

As seen from above Table 1 the Isomaltooligosaccharide has a MW of 1020Da and Mn equals 827 Da giving a polydispersity Pd=1.23. The reducingsugar is measured to be 22.5%. This isomaltooligosahharide is also namedpentaisomaltose in this application.

Preparation Example 2 Production of Hydrogenated Isomaltooligosaccharide1

After hydrolysis and fractionation 418 kg isomaltooligosaccharide wasleft. The reducing sugar was measured to 30.8%. This amount was treatedwith 10 kg Sodium borohydride and gave as a result 362 kg hydrogenatedisomaltooligosaccharide before final ion exchange. Hereafter thesolution was neutralized to pH<7.0, and subsequently de-ionized andfinally spray dried. The reducing sugar of the final product wasmeasured to 0.09%.

TABLE 2 Results from GPC analysis. Number of glucose RelativeContribution Relative Contribution units AUPW AUPW Mi to M_(W) AUPN AUPNto M_(n) 1 0.31 0.0033 180 1 0.0017 0.0145 3 2 4.38 0.0472 342 16 0.01280.1076 37 3 14.55 0.1568 504 79 0.0289 0.2425 122 4 16.47 0.1775 666 1180.0247 0.2077 138 5 14.65 0.1579 828 131 0.0177 0.1486 123 6 11.570.1247 990 123 0.0117 0.0982 97 7 8.61 0.0928 1152 107 0.0075 0.0628 728 6.57 0.0708 1314 93 0.0050 0.0420 55 9 4.77 0.0514 1476 76 0.00320.0271 40 10 3.43 0.0370 1638 61 0.0021 0.0176 29 11 2.43 0.0262 1800 470.0014 0.0113 20 12 1.76 0.0190 1962 37 0.0009 0.0075 15 13 1.19 0.01282124 27 0.0006 0.0047 10 14 2.11 0.0227 2286 52 0.0009 0.0078 18 92.801.0000 968 0.1190 1.0000 780 AUPW: Area under peak (M_(w)) AUPN: Areaunder peak (M_(n))

As seen from above table 2 the hydrogenated isomaltooligosaccharide hasa M_(w) of 968 Da and M_(n) equals 780 Da giving a polydispersityPd=1.24. This hydrogenated isomaltooligosahharide is also namedpentaisomaltoside in this application.

Example 1 Preparation of Cryopreservation Agent

The cryopreservation agent used in the following examples were preparedby aseptically solubilizing the cryoprotectant (such as DMSO,pentaisomaltose (isomaltooligosaccharide 1) prepared as described inPreparation Example 1 or pentaisomaltoside (hydrogenatedisomaltooligosaccharide 1) prepared as described in Preparation Example2) in growth medium (DMEM/F12+10% FBS+penicillin/streptomycin) to thedesired final concentration (e.g. for 8% isomaltooligosaccharide 1cryopreservation composition, 8 gram to 100 ml growth medium) and filtersterilize the individual cryopreservation compositions.

Example 2 Cryopreservation of Normal Human Dermal Fibroblasts (NHDF)

NHDF (passage 2) were cultured in conventional T-flasks under standardconditions (37° C., 5% CO₂ and standard growth medium [DMEM/F12+10%FBS+penicillin/streptomycin)]). When reaching confluence (70-80%) thecell population was released from the flasks and centrifuged (1000 rpm,10 min). 0.5×10⁶ cells were re-suspended in six differentcryopreservation agents (1 ml); 1) Growth medium+10% DMSO, 2) Growthmedium+2% DMSO+8% isomaltooligosaccharide 1, 3) Growth medium+2% DMSO+8%hydrogenated isomaltooligosaccharide 1, 4) Growth medium+8%isomaltooligosaccharide 1, 5) Growth medium+8% hydrogenatedisomaltooligosaccharide 1 and 6) Growth medium without any additives(DMEM/F12 without FBS). The cells were then cryopreserved under standardcontrolled cryopreservation conditions using isopropanol basedmethodology, freezing at the constant rate 1° C./min down to thetemperature of liquid N₂. After one week the cells were thawed using astandard thawing protocol (directly immerse vial in water bath, 37degrees and transfer of cell solution drop wise to fresh 37° C. growthmedium). The following analysis was done after thawing; 1) viability,using the Nucleocounter equipment (NC200), and 2) viability at 1.passageusing Nucleocounter equipment (NC200). The results are shown in FIGS. 1and 2 and summarized in table 3-5.

The viability after thawing is slightly reduced when usingisomaltooligosaccharide 1 as the only cryopreservation agent as comparedto standard condition using 10% DMSO. When using 2% DMSO together withisomaltooligosaccharide 1 no difference with regard to viability isobserved. The viability of cells cryopreserved withisomaltooligosaccharide 1 alone at 1.passage is similar to standardconditions using 10% DMSO. This experiment demonstrates thatcryopreserving NHDF in a cryopreservation solution usingisomaltooligosaccharide 1 as the only cryoprotectant results in culturesthat can be used to the same extent as cultures from standardcryopreservation conditions.

Example 3 Cryopreservation of Normal Human Keratinocytes (NHEKs)

NHEKs were cryopreserved using the same protocol as described in Example2. The same experimental groups were included. Results are shown inFIGS. 3-4 and are summarized in table 3-5.

As also shown for NHDF, the results clearly demonstrate that NHEKs canbe cryopreserved in a cryopreservation solution usingisomaltooligosaccharide 1 as the only cryoprotectant. The viability atfirst passage of culture is at the same level as NHEKS cryopreservedunder standard conditions using 10% DMSO.

Example 4 Cryopreservation of Normal Human Mesenchymal Stem Cells(hMSCs)

hMSCs were cryopreserved using the same protocol as described in Example2 with two more experimental groups included, growth medium+8%isomaltooligosaccharide 1+2% trehalose and growth medium+2% trehalose.Viability analysis was done, using the Nucleocounter technique asdescribed above. The results are shown in FIG. 5 and summarized in table3-5.

The results clearly demonstrate that hMSCs can be cryopreserved in acryopreservation solution using isomaltooligosaccharide Da as the onlycryoprotectant. The viability after thawing is shown to be at the samelevel as the standard formulation containing 10% DMSO.

Table 3-5:

T1: Survival after thawing

T2: Survival after 1.passage

TABLE 3 10% NHDF NHEK hMSC DMSO T1 T2 T1 T2 T1 T2 10% 96 ± 5% 92 ± 2% 92± 3% 88 ± 84 ± 10% N/A DMSO 5% 10% 98 ± 2% 95 ± 2% 75 ± 4% N/A 90 ± 2% N/A DMSO

TABLE 4 NHDF NHEK hMSCs T1 T2 T1 T2 T1 T2 2% DMSO + 8% 96 ± 5% 92 ± 2%93 ± 2% 96 ± 1% 95 ± 1% N/A isomaltooligosaccharide 1 2% DMSO + 8% 98 ±1% 96 ± 1% 81 ± 5% N/A 90 ± 3 N/A Hydrogenated isomaltooligosaccharide 1

TABLE 5 NHDF NHEK hMSC T1 T2 T1 T2 T1 T2 8% 75 ± 5% 85 ± 5% 74 ± 1%  85± 8% 80 ± 7% N/A isomaltooligosaccharide 1 8% Hydrogenated 82 ± 5% 90 ±3% 85 ± 11% N/A 80 ± 5 N/A isomaltooligosaccharide 1

Example 5 Exposure of hMSCs to Isomaltooligosaccharide 1

hMSCs were grown to confluence in conventional T-flasks. Cells werereleased and resuspended in two different formulations 1) growthmedium+10% DMSO and 2) 8% isomaltooligosaccharide 1. The finalconcentration of cells in each formulation was of 1×10⁶/ml cells. Thesame basic protocol as described in Example 2 was used. 1 ml from eachvial were added to cryovials and viability was analysed using theNucleoCounter at three different time points; 1) 0 min (TO), 10 min(T10) and 30 min (T30). Results are summarized in table 6.

TABLE 6 T0 (%) T10 (%) T30 (%) 10% DMS0 100 67 41 8% 100 97 95isomaltooligosaccharide 1

It is clearly demonstrated that exposure to standard cryopreservationconditions seriously affects the viability of hMSCs. Exposure to thecryoformulation with isomaltooligosaccharide 1 as the onlycryoprotectant component only affects the viability significantly afterexposure for 60 min. This demonstrates that it is possible to handlecell cultures for cryopreservation in the cryopreservation compositioncontaining isomaltooligosaccharide 1, making more flexible workingprocedures possible.

Example 6 Isomaltooligosaccharide 560 Da for Cryopreservation of hMSCs

hMSCs were cryopreserved using the same basic protocol as described inExample 2 and the same experimental groups were included. The resultsdemonstrate that it is possible to cryopreserve hMSCs inisomaltooligosaccharide 560 Da, however hydrogenatedisomaltooligosaccharide 560 Da is not as effective asisomaltooligosaccharide 560 Da in this experiment. Results aresummarized in table 7.

TABLE 7 8% 8% 8% Hydrogenated 8% Hydrogenated 10%isomaltooligosaccharide isomaltooligosaccharide isomaltooligosaccharideisomaltooligosaccharide DMSO 560 Da + 2% DMSO 560 Da 560 Da + 2% DMSO560 Da 90 ± 1% 66 ± 15% 78 ± 1% 60 ± 1% 44 ± 5%

Example 7 Human iPS Cells in PluriPro Growth Medium

The isomaltooligosaccharide 1 used in this example is prepared asdescribed in Preparation example 1. Human induced pluripotent stemcells, iPSCs, (passage 12) were cultured in conventional T-flasks assingle cells under standard conditions (37 degrees, 5% CO₂ and PluriProgrowth medium, Cell Guidance System). When reaching confluence (70-80%)the cell population was released from the flasks and centrifuged (1000rpm, 10 min). 0.5×10⁶ cells were resuspended in twelve differentcryopreservation solutions (1 ml); Growth medium+10% DMSO, Growthmedium+5% DMSO, Growth medium+10% DMSO+2% isomaltooligosaccharide 1,Growth medium+10% DMSO+4% isomaltooligosaccharide 1, Growth medium+10%DMSO+8% isomaltooligosaccharide 1, Growth medium+5% DMSO+2%isomaltooligosaccharide 1, Growth medium+5% DMSO+4%isomaltooligosaccharide 1, Growth medium+5% DMSO+8%isomaltooligosaccharide 1, Growth medium+2% isomaltooligosaccharide 1,Growth medium+4% isomaltooligosaccharide 1, Growth medium+8%isomaltooligosaccharide 1, and Growth medium without any cryoprotectant.The cells were then cryopreserved under standard controlledcryopreservation conditions (using an isopropanol based methodology)down to liquid N₂. After one week the cells were thawed using standardthawing protocol (directly immerse vial in water bath, 37 degrees andtransfer of cell solution dropwise to fresh growth medium). ROCKinhibitor was added under the first seeding. Viability analysis afterthawing was done, using the Nucleocounter technique. The results areshown in FIG. 6.

The results demonstrate that human iPS cells can be cryopreserved usingisomaltooligosaccharide 1 as the only cryoprotectant, although theviability of the cryopreserved cells is significantly lower than caseswhere DMSO is added.

Example 8 Human iPS Cells in PluriPro Growth Medium

The hydrogenated isomaltooligosaccharide 1 used in this example isprepared as described in Preparation example 2. Human inducedpluripotent stem cells, iPSCs, (passage 12) were cultured inconventional T-flasks as single cells under standard conditions (37degrees, 5% CO₂ and PluriPro growth medium, Cell Guidance System). Whenreaching confluence (70-80%) the cell population was released from theflasks and centrifuged (1000 rpm, 10 min). 0.5×10⁶ cells wereresuspended in twelve different cryopreservation solutions (1 ml);Growth medium+10% DMSO, Growth medium+5% DMSO, Growth medium+10% DMSO+2%hydrogenated isomaltooligosaccharide 1, Growth medium+10% DMSO+4%hydrogenated isomaltooligosaccharide 1, Growth medium+10% DMSO+8%hydrogenated isomaltooligosaccharide 1, Growth medium+5% DMSO+2%hydrogenated isomaltooligosaccharide 1, Growth medium+5% DMSO+4%hydrogenated isomaltooligosaccharide 1, Growth medium+5% DMSO+8%hydrogenated isomaltooligosaccharide 1, Growth medium+2% hydrogenatedisomaltooligosaccharide 1, Growth medium+4% hydrogenatedisomaltooligosaccharide 1, Growth medium+8% hydrogenatedisomaltooligosaccharide 1 and Growth medium without any cryoprotectant.The cells were then cryopreserved under standard controlledcryopreservation conditions (using an isopropanol based methodology)down to liquid N₂. After one week the cells were thawed using standardthawing protocol (directly immerse vial in water bath, 37 degrees andtransfer of cell solution dropwise to fresh growth medium). ROCKinhibitor was added under the first seeding. Viability analysis afterthawing was done, using the Nucleocounter technique. The results areshown in FIG. 7.

The results demonstrate that human iPS cells can be cryopreserved usinghydrogenated isomaltooligosaccharide 1 as the only cryoprotectant,although the viability of the cryopreserved cells is significantly lowerthan cases where DMSO is added. In the samples cryopreserved withoutDMSO, a tendency to improved viability as a function of usedIsomaltooligosaccharide 1 concentration was observed.

Example 9 Cryopreservation of Normal Human Mesenchymal Stem Cells(hMSCs) in Cryoprotectants with Different Molecular Weights

hMSCs were cryopreserved using the same protocol as described in Example2 involving the following experimental groups, growth medium+10% DMSO,8% isomaltooligosaccharide 1, Dextran average Mw 10.000 or Dextranaverage Mw 40.000+5% DMSO, 8% isomaltooligosaccharide 1, Dextran averageMw10.000 or Dextran average Mw 40.000+1% DMSO, 8%isomaltooligosaccharide 1, Dextran average Mw10.000 or Dextran averageMw 40.000 and growth medium. Viability analysis was done, using theNucleocounter technique as described above. After thawing MSCs werecultured for 3 days under standard conditions and the proliferative ratewas analysed by using colorimetric in vitro analysis, MTT assay thatmeasures the mitochondrial activity in the cell population.

The results are shown in FIG. 8.

The results clearly demonstrate that hMSCs can be cryopreserved in acryopreservation solution using isomaltooligosaccharide 1, Dextranaverage Mw 10.000 or Dextran average Mw 40.000 as the onlycryoprotectant. No significant difference was observed between Mw on thedirect viability analysis after thawing. However the analysis of theproliferative rate after 3 days demonstrated that hMSCs cryopreserved in8% isomaltooligosaccharide 1 (average Mw 1000) proliferated moreactively compared to 8% Dextran average Mw 10.000 and average Mw 40.000.

Example 10 Cryopreservation of Normal Human Mesenchymal Stem Cells(hMSCs) in Isomaltooligosaccharide Having an Average Mw 1500 Mw

hMSCs were cryopreserved using the same protocol as described in Example2 involving the following experimental groups, growth medium+10% DMSO,growth medium+2% DMSO, 8% isomaltooligosaccharide having an average Mw1500 Mw+2% DMSO, 8% isomaltooligosaccharide having an average Mw 1500 Mwand growth medium. Viability analysis was done, using the Nucleocountertechnique as described above. The results are shown in FIG. 9.

The results clearly demonstrate that hMSCs can be cryopreserved in acryopreservation solution using isomaltooligosaccharide having anaverage Mw 1500 as the only cryoprotectant.

Example 11 Viability of CD34⁺ Hematopoietic Stem Cell FollowingCryopreservation with DMSO, Isomaltooligosaccharide 1 or HydrogenatedIsomaltooligosaccharide 1

Mobilized peripheral blood cells were harvested by leukapheresis andfrozen in cryoprotective medium containing 10% DMSO or differentconcentrations of isomaltooligosaccharide 1 (isom) or hydrogenatedisomaltooligosaccharide 1 (h-isom). Samples were frozen using acontrolled rate freezer (Kryo 560-16, Planer; start temp. 4° C., −1°C./min drop to 0° C., −2° C./min drop to −45° C. and −5° C./min drop to−100° C.) and moved to −150° C. Samples were thawed in a 37° C. waterbath. Flow cytometry was applied to estimate the viability of CD45+,CD34+ hematopoietic stem cells. The fluorescent DNA binding compound7-Aminoactinomycin D (7-AAD) was used as a live/dead marker. Cells ableto exclude 7AAD were assumed to be viable. The results are shown in FIG.10: Isomaltooligosaccharide 1 (light grey bars), hydrogenatedisomaltooligosaccharide 1 (dark grey bars) and DMSO (black bars). Datashown here are from 3 separate experiments, each measured in doublets.Error bars indicate the standard deviation.

Both h-isom and isom supports the viability of CD34+ hematopoietic cellsafter cryopreservation to the same extent as standard 10% DMSO. A cleartendency towards a higher protective effect with higher concentrationsof both i-som and isom is demonstrated. Concentrations of 4% exert asignificant lesser protective effect (below 60%) than concentrations of6%_(,) 8%, 10% and 12%. Concentrations of 10% and 12% have similarprotective effect as compared to 10% DMSO. No significant differencebetween the protective effects of h-isom and isom was observed in thestudy.

Example 12 Viability of Adipose-Derived Stromal/Stem Cells (ASC's)Following Cryopreservation with DMSO, Isomaltooligosaccharide 1 orHydrogenated Isomaltooligosaccharide 1

ASC's were harvested from adipose tissue obtained by cosmeticliposuction of the abdomen or the inner thighs using the Vibrasat device(Möller Medical GmbH & Co. KG, Fulda, Germany). The ASC's from thestromal vascular fraction were expanded ex vivo in culture mediumconsisting of Dulbecco's modified Eagle's medium, 1%penicillin-streptomycin, 1% GlutaMAX, and 10% pooled human plateletlysate. Cells were frozen in cryoprotective medium containing 10% DMSOor different concentrations of isomaltooligosaccharide 1 (isom) orhydrogenated isomaltooligosaccharide 1 (h-isom). Samples were frozenusing a controlled rate freezer (Kryo 560-16, Planer; start temp. 4° C.,−1° C./min drop to 0° C., −2° C./min drop to −45° C. and −5° C./min dropto −100° C.) and moved to −150° C. Samples were thawed in a 37° C. waterbath. Flow cytometry was applied for phenotype characterization(positive for CD73, CD90, CD105 and negative for CD14, CD20, CD45 andCD34) and to estimate the viability of ASC's. The fluorescent DNAbinding compound 7-Aminoactinomycin D (7-AAD) was used as a live/deadmarker. Cells able to exclude 7AAD were assumed to be viable. Theresults are shown in FIG. 11: Isomaltooligosaccharide 1 (light greybars), hydrogenated isomaltooligosaccharide 1 (dark grey bars) and DMSO(black bars). Data shown here are from a single experiment measured indoublets. Error bars indicate the standard deviation. The result forDMSO is lower than expected.

Both h-isom and isom as cryoprotectants supports the viability ASCs tothe same extent as 10% DMSO. Except for low concentration of 4%, higherconcentrations results in a viability above 70%. A concentration of 12%results in a significantly higher viability (80%-90%) compared to lowerconcentrations. No significant difference between the cryoprotectiveeffects of h-isom and isom was observed in the study except for the 4%group.

Example 13

After removal of mouse ovaries, they are transferred to a McCoy mediumsupplemented with 10 mg/ml HSA, pen/strep and kept at 37° C. until theyare transferred to below cryopreservation solutions: 1) standardconditions (PBS, 1.5 mol/L ethylene glycol, 0.1 mol/L sucrose, 10 mg/mlHSA similar to cryopreservation of human ovarian tissue) or 2) with PBS,10° A) (w/v) Isomaltooligosaccharide 1.

The ovaries are equilibrated for 30 min on ice and then transferred to aprogrammable cryofreezer (Planner Cryo 10 programmable freezer, UK)where the samples are cooled to −140° C. with the following ramps (starttemp: −1° C.; −2° C./min until −9° C.; holding 5 min; seeding; −0.3°C./min until −40° C., −10° C./min until −140C and then directly inliquid nitrogen). After this, they are plunged into liquid nitrogen andkept in a liquid nitrogen tank for a variable time period. Thawing: roomtemperature in 37C hot water; 10 min a medium withIsomaltooligosaccharide 1 (20° A) (w/v) and then directly into fixationmedium. On histological preparations both ovaries show survivingfollicles in different stages of development.

Example 14

Mouse ovaries are treated as described in above Example 13 but in belowcryopreservation solutions: 1) standard conditions (PBS, 1.5 mol/Lethylene glycol, 0.1 mol/L sucrose, 10 mg/ml HSA similar tocryopreservation of human ovarian tissue) or 2) with PBS, 1.5 mol/Lethylene glycol, 10 mg/ml HSA, 10° A) (w/v) hydrogenatedisomaltooligosaccharide 1 or 3) with PBS, 10 mg/ml HSA, 10° A) (w/v)hydrogenated isomaltooligosaccharide 1. On histological preparations allovaries show surviving follicles in different stages of development.

The invention claimed is:
 1. A method of protecting a sample fromfreezing damage, comprising the steps of: (i) bringing a samplecomprising peripheral blood derived hematopoietic stem cells to becryopreserved into contact with a cryoprotective medium comprising acryoprotecting agent to obtain a cryopreservation composition; (ii)subsequently reducing the temperature of the cryopreservationcomposition to a cryopreservation temperature, where said cryoprotectingagent comprises one or more cryoprotectants selected fromisomaltooligosaccharide and hydrogenated isomaltooligosaccharide,wherein said one or more cryoprotectants has a weight average molecularweight (Mw) of between 850 and 1,150 Da, and the cryoprotectivecomposition is substantially free of dimethyl sulfoxide (DMSO).
 2. Themethod according to claim 1, wherein at least one of the one or morecryoprotectants has a polydispersity of ≥1 and ≤5.
 3. The methodaccording to claim 1, wherein said one or more cryoprotectants is anisomaltooligosaccharide having a weight average molecular weight (Mw) ofbetween 850 and 1,150 Da.
 4. The method according to claim 1, whereinsaid one or more cryoprotectants is an hydrogenatedisomaltooligosaccharide having a weight average molecular weight (Mw) ofbetween 850 and 1,150 Da.
 5. The method according to claim 1, whereinthe weight fraction of isomaltooligosaccharides in the cryoprotectivemedium having less than 3 glucose units is less than 15% w/w and/orwherein the weight fraction of isomaltooligosaccharides in thecryoprotective medium having more than 9 glucose units is less than 20%w/w.
 6. The method according to claim 1, wherein saidisomaltooligosaccharides and hydrogenated isomaltooligosaccharides aredextran-based.
 7. The method according to claim 1, wherein saidcryoprotecting agent is in the form of a powder or in the form of asolution.
 8. The method according to claim 1, wherein saidcryoprotecting agent comprises from 30% to 70% w/w of said one or morecryoprotectants.
 9. The method according to claim 1, wherein saidcryoprotecting agent comprises an additional cryoprotectant, whereinsaid additional cryoprotectant is ethylene glycol.
 10. The methodaccording to claim 1, wherein said cryoprotecting agent comprises anadditional cryoprotectant, wherein said additional cryoprotectant isalbumin.
 11. The method according to claim 1, wherein the sampleconsists of hematopoietic stem cells.
 12. The method of claim 1, whereinthe peripheral blood derived hematopoietic stem cells are CD34 positiveblood stem cells.
 13. The method of claim 1, further comprising (iii)thawing the sample after cryopreservation to generate a thawed sample;(iv) directly suspending the thawed sample in a culture medium; and (v)culturing the thawed sample in the culture medium, wherein the methoddoes not include washing the thawed sample prior to suspending or priorto culturing the thawed sample in the culture medium.
 14. The methodaccording to claim 1, wherein said sample comprises in vitro culturedhematopoietic stem cells.
 15. The method according to claim 1, whereinsaid sample is suitable for use in hematopoietic stem cellstransplantation.
 16. A method of protecting a sample from freezingdamage, comprising the steps of: (i) bringing a sample comprisingperipheral blood derived hematopoietic stem cells to be cryopreservedinto contact with a cryoprotective medium comprising a cryoprotectingagent to obtain a cryopreservation composition; (ii) subsequentlyreducing the temperature of the cryopreservation composition to acryopreservation temperature, where said cryoprotecting agent comprisesone or more cryoprotectants selected from isomaltooligosaccharides andhydrogenated isomaltooligosaccharides, wherein said one or morecryoprotectants has a weight average molecular weight (Mw) of between850 and 1,150 Da, the cryoprotective composition is substantially freeof dimethyl sulfoxide (DMSO), and said cryoprotecting agent furthercomprises an additional cryoprotectant, which additional cryoprotectantis albumin, wherein said cryoprotective medium comprises no more than 8%w/w albumin.
 17. The method according to claim 16, wherein saidcryoprotective medium comprises from 1-8% albumin.
 18. The methodaccording to claim 16, wherein said cryoprotective medium comprises nomore than 5% w/w albumin.
 19. The method according to claim 16, whereinsaid cryoprotective medium comprises from 1-4% w/w albumin.
 20. Themethod according to claim 16, wherein at least one of said one or morecryoprotectants has a polydispersity of ≥1 and ≤5.
 21. The methodaccording to claim 16, wherein said cryoprotectants selected fromisomaltooligosaccharides and hydrogenated isomaltooligosaccharidesconsist of isomaltooligosaccharides having a weight average molecularweight (Mw) of between 850 and 1,150 Da.
 22. The method according toclaim 16, wherein said cryoprotectants selected fromisomaltooligosaccharides and hydrogenated isomaltooligosaccharidesconsist of hydrogenated isomaltooligosaccharides having a weight averagemolecular weight (Mw) of between 850 and 1,150 Da.
 23. The methodaccording to claim 16, wherein the weight fraction ofisomaltooligosaccharides in the cryoprotective medium having less than 3glucose units is less than 15% w/w and/or wherein the weight fraction ofisomaltooligosaccharides in the cryoprotective medium having more than 9glucose units is less than 20% w/w.
 24. The method according to claim16, wherein said isomaltooligosaccharides and hydrogenatedisomaltooligosaccharides are dextran-based.
 25. The method according toclaim 16, wherein said cryoprotecting agent is in the form of a powderor in the form of a solution.
 26. The method according to claim 16,wherein said cryoprotecting agent comprises from 30% to 70% w/w of saidone or more cryoprotectants.
 27. The method according to claim 16,wherein the sample consists of hematopoietic stem cells.
 28. The methodaccording to claim 16, wherein the peripheral blood derivedhematopoietic stem cells are CD34 positive blood stem cells.
 29. Themethod according to claim 16, further comprising (iii) thawing thesample after cryopreservation to generate a thawed sample; (iv) directlysuspending the thawed sample in a culture medium; and (v) culturing thethawed sample in the culture medium, wherein the method does not includewashing the thawed sample prior to suspending or prior to culturing thethawed sample in the culture medium.
 30. The method according to claim16, wherein said sample comprises in vitro cultured hematopoietic stemcells.
 31. The method according to claim 16, wherein said sample issuitable for use in hematopoietic stem cells transplantation.